EXTRACT OF AN HERBAL COMPOSITION AS ANTIMICROBIAL AND/OR ANTIBIOFILM AGENT

- ALPHANOSOS S.A.S.

An extract of an herbal composition comprising at least two different dried plants useful as antimicrobial and/or antibiofilm agent in the treatment or prevention of microbial infections caused by bacteria, such as for example Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Streptococcus or Staphylococcus, or by fungi, such as for example is herein described. It has been found that in such extract, the active ingredients exert their biological effects in a synergistic manner. The extract may constitute the active ingredient of a food supplement, a nutraceutical, pharmaceutical or cosmetic composition or a functional food or a food additive. A process for preparing said extract is also described here.

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Description

This application is a Continuation-in-Part of U.S. Ser. No. 16/345,844 filed Apr. 29, 2019, which is a National Stage of PCT/EP2017/077956 filed on 1 Nov. 2017, which claims priority to and the benefit of European Application No. 16197217.9 filed on 4 Nov. 2016, and which claims priority to and the benefit of French Application no. 1660588 filed on 2 Nov. 2016, the contents of which are all incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to an extract of an herbal composition comprising at least two different dried plants useful as antimicrobial and/or antibiofilm agent. It has also been found that in such extract, the active ingredients exert their biological effects in a synergistic manner.

BACKGROUND OF THE INVENTION

Like any living organism, plants contain numerous chemical entities, comprising low molecular weight chemicals, polypeptides, polysaccharides, enzymes. A plant extract is thus a trivial example of naturally occurring mix of chemical entities.

The biological and therapeutic activities of many chemical entities mixes obtained from plants is of secular knowledge and is compiled in numerous manuscripts (see for example, Encyclopedia of Herbal Medicine: The Definitive Home Reference Guide to 550 Key Herbs with all their Uses as Remedies for Common Ailments Hardcover—Nov. 15, 2000, by Andrew Chevallier).

Modern science allows to observe biological and therapeutic effects of chemical entities combinations obtained from plants on a large number of organisms.

For instance, it is described in Scheggi (Scheggi S. et al, Pharm. Biol. 2016 Jan. 5:1-11. [Epub ahead of print]) how a rat model allows to test the effect against depression of chemical entities combinations obtained from plants.

In Zhipeng Q U (Oncotarget. 2016 Sep. 1 Epub) it is shown how it is possible to identify candidate anti-cancer molecular mechanisms of Compound Kushen Injection using functional genomics. Compound Kushen Injection (CKI) has been clinically used in China for over 15 years to treat various types of solid tumours. However, because such Traditional Chinese Medicine (TCM) preparations are complex mixtures of plant secondary metabolites, it is essential to explore their underlying molecular mechanisms in a systematic fashion. This study shows that CKI inhibited MCF-7 cell proliferation and induced apoptosis in a dose-dependent fashion. Multiple pathways were found to be perturbed and the cell cycle was identified as the potential primary target pathway of CKI in MCF-7 cells. CKI may also induce apoptosis in MCF-7 cells via a p53 independent mechanism. In addition, they identified novel lncRNAs and showed that many of them might be expressed as a response to CKI treatment.

The effect of chemical entities mixtures obtained for instance from plants, fungi or bacteria on the growth of microorganisms such as bacteria and fungi has been described many times, for instance in Venkatadri (Venkatadri B. et al., Indian J. Pharm. Sci. 2015 November-December; 77(6):788-91).

The effect of chemical entities combinations obtained for instance from plants, fungi or bacteria on the formation of biofilms (communities of microorganisms embedded in a polymeric matrix attached to a surface) has also been described many times, for instance in Suntar (Suntar I. et al., Pharm. Biol. 2016 June; 54(6):1065-70Epub 2015 Oct. 29).

Shimizu M. et al. (Antimicrob Agents Chemother. 2001 November; 45(11):3198-201) found that an extract of Arctostaphylos uva-ursi markedly reduced the MICs (Minimum Inhibitory Concentrations) of beta-lactam antibiotics, such as oxacillin and cefmetazole, against methicillin-resistant Staphylococcus aureus. The same group also isolated the effective compound and identified it as corilagin.

Another group (Denev P. et al., Acta Biochim. Pol. 2014; 61(2):359-67. Epub 2014 Jun. 18) studied the antioxidant, antimicrobial and neutrophil-modulating activities of extracts from six medicinal plants—blackberry (Rubus fruticosus) leaves, chokeberry (Aronia melanocarpa) leaves, hawthorn (Crataegus monogyna) leaves, lady's mantle (Alchemilla glabra) aerial parts, meadowsweet (Filipendula ulmaria) aerial parts and raspberry (Rubus idaeus) leaves. The antimicrobial activity of the investigated extracts against 11 human pathogens was investigated using three different methods and the results of this study allowed concluding that meadowsweet and blackberry leaves extracts had the highest antimicrobial effect and the lowest minimal inhibiting concentrations (MICs) against the microorganisms tested.

Yam T. et al. (FEMS Microbiol Lett. 1997 Jul. 1; 152(1):169-74) focused their study on the microbiological activity of whole and fractionated crude extracts of tea (Camellia sinensis), and of tea components. Aqueous extracts of teas (Camellia sinensis) of different types and from various sources were tested and found to inhibit a wide range of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus. Tea extracts were bactericidal to staphylococci and Yersinia enterocolitica at well below ‘cup of tea’ concentrations. Activity was confined to one of four fractions obtained from a green tea extract by partition chromatography. Testing of pure tea compounds and closely related chemicals suggested that the antibacterial activity of extracts of green tea can be explained by its content of epigallocatechin, epigallocatechin gallate and epicatechin gallate. In black tea extracts, theaflavin and its gallates are additional antibacterially active components.

WO 2009/031041 reports a composition comprising (a) an antimicrobial compound having a given chemical structural formula, (b) an antimicrobial material selected from lanthionine bacteriocins, tea [Camellia sinensis] extract, hop [Humulus lupulus L] extract, grape skin extract, grape seed extract, Uva Ursi [Arctostaphylos uva-ursi] extract and combinations thereof.

WO 99/20289 reports a clear herbal extract solution comprising a concentrated herbal extract and a fill liquid. The concentrated herbal extract is reported to be selected from a long list of plants extracts, where such list includes Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi. However, WO 99/20289 does not mention any specific therapeutic or medical use of the claimed herbal solution.

US 2015/0056255 reports a product for medicinal, cosmetic, coloring, or dermatologic use, the product comprising a layer of a fibrous plant product and a plant extract applied to the layer of the fibrous plant product. The plant extract is said to comprise substances from one or more specific parts of one or more plants, wherein the plants are selected from at least one of a long list of plants.

Azanida N N (Med. Aromat. Plants, 4:3, p. 1-6, 2015) published a review, which reports a description and a comparison of the most commonly used methods for preparing medicinal plant extracts. The authors conclude their article by stating that “no universal extraction method is the ideal method and each extraction procedure is unique to the plants” (see “Conclusions”, lines 5-7).

Selman A. Wachsman et al. (Bacteriology, 31, p. 157-164, 1945) reports that different strains of the same bacterial species may vary greatly in their resistance to streptomycin. Different Staphylococcus aureus bacterial strains are used in such paper to test the activity of streptomycin. However, no plant extract is tested on the same strains and therefore no clear guidance can be drawn from such paper.

Biological effects such as contemplated in the previous examples can be induced either by a single chemical entity, such as in standard drugs, or by a synergistic effects of different chemical entities, such as in extracts. In some cases, the biological effect is nearly fully attributable to a single chemical entity contained in the extract. In this case, the extract can be replaced by the purified active chemical entity.

Biological effects of a chemical entity can be either due to chemical reactions (transformations) with the chemical entities constituting the organism or be due to the transient or permanent binding to the chemical entities, in particular the macromolecules, constituting the organism. Such binding will modulate the activity of the organism's chemical entities, in particular the macromolecules, e.g., increase or decrease the activity of an enzyme.

Unfortunately, chemical entities have never a single effect on an organism. In particular, the interactions with the macromolecular constituents are numerous. As a matter of fact, it is well established that any chemical entity, whether natural or resulting from chemical synthesis, will have deleterious effects on organisms (humans, animals, plants, micro-flora, etc.) in a dose related fashion. Eventually, a dose related positive or therapeutic effect can be observed also. It is obvious that for the chemical entity to be of industrial interest, the dose required to observe the positive or therapeutic effect needs to be lower than the dose for which the deleterious effects become unacceptable.

Chemical entities differing in their chemical composition or structure will have different affinities (or binding constants) with the different macromolecules constituting the organism, and thus different modes of actions resulting in the observed deleterious, positive and therapeutic effects.

The macromolecules involved in deleterious effects and those involved in positive or therapeutic effects are different in many cases. Chemical entities differing in their chemical composition will in most of the cases have very different affinities (or binding constants) with the numerous different macromolecules constituting the organism.

In most cases, different chemical entities will have different affinities (or binding constants) with a given macromolecule. In rare cases, different chemical entities will have similar affinities (or binding constants) with the same macromolecule or sets of macromolecules involved in a positive or therapeutic effect. Among these cases, it will be rare that these different chemical entities will have comparable affinities (or binding constants) with the macromolecules involved in given deleterious effect.

Mixes of different chemical entities may thus have a synergistic positive or therapeutic effect without a cumulative given deleterious effect. Thus, for a given positive or therapeutic effect, the dose required for each individual different chemical entity in a mix will be lower than the dose required by each of the chemical entity considered individually to obtain that given positive or therapeutic effect. In those cases where the deleterious effects induced by each different chemical compound is different or has a different macromolecular aetiology, each deleterious effect of the mix will be lower than the deleterious effect of the chemical entity considered individually because it is used at a lower dose in the mix.

Note that the synergistic situation of the simultaneous administration of reduced doses of different chemical entities is different from the co-administration of different drugs (chemical entities) having different therapeutic effects administered at their standard doses. In the latter situation, increased side effects due to drug-interactions are well known. One of the causes of side effects is the possible chemical interaction of the different drugs (chemical entities). In the synergistic situation, each chemical entity is present at a reduced concentration and thus with much lower rates of chemical interactions. For illustration purposes, a synergistic mix of 10 different chemical entities (drugs) administrated each at 1/10th of the concentration (dose) required for a chemical entity (drug) administrated individually, the rate of inter-drug chemical reactions will be reduced by a factor of 100 as implied by the universal law of action of mass.

It is therefore of interest to use synergistic mixes of different chemical entities in order to obtain a given positive or therapeutic effect with reduced deleterious effects, or to obtain an increased positive or therapeutic effect with a given level of different deleterious effects. Indeed, several extracts are known have synergistic activities relative to isolated chemical entities (see for instance, Deharo E. et al., Malar. J. 2011 Mar. 15; 10 Suppl 1:55).

Natural extracts composed of a mix of synergistic chemical entities can be discovered for example by means of systematic testing numerous natural extracts of single plant powders or herbal compositions containing at least two or more different plant powders.

DESCRIPTION OF THE INVENTION

It has now been found that an extract of an herbal composition comprising at least two dried plants selected among Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi is useful as antimicrobial and/or antibiofilm agent in the treatment or prevention of microbial infections caused by bacteria, such as for example Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Streptococcus or Staphylococcus, or by fungi, such as for example Candida. It has also been found that in such extract, the active ingredients exert their biological effects in a synergistic manner.

Therefore, one object of the present invention is a method for the treatment or prevention of the above mentioned microbial infections comprising administering an effective amount of an extract of an herbal composition comprising at least two dried plants selected among Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi, a lyophilisate of said extract, or a pharmaceutical or veterinary composition comprising said extract or said lyophilisate, to a subject in the need thereof.

Another object of the present invention is an extract of an herbal composition, comprising at least two dried plants selected among Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi and at least one plant selected among Vitis vinifera var. tinctoria, Eugenia caryophyllus and Desmodium adscendens.

Another object of the present invention is an extract of an herbal composition comprising the following dried plants: Filipendula ulmaria, Camellia sinensis and Vitis vinifera var. tinctoria, wherein the w/w weight ratio between Filipendula ulmaria and each of the dried Camellia sinensis and Vitis vinifera var. tinctoria is between 0.2 and 5, optionally between 0.3 and 3, more optionally between 0.5 and 2.5.

Said herbal composition may further comprise one or more additional dried plants selected among:

Achillea millefolium, Acorus calamus, Agrimonia eupatoria, Agropyrum repens, Agropyrum repens, Alchemilla vulgaris, Alkanna tinctoria, Althaea officinalis, Anethum graveolens, Angelica archangelica, Arbutus unedo, Arnica montana, Artemisia pontica, Artemisia vulgaris, Asparagus officinalis, Asparagus officinalis, Asperula odorata, Betula pendula, Borrago officinalis, Buxus sempervirens, Calamintha officinalis, Calendula officinalis, Calluna vulgaris, Carum carvi, Cassia angustifolia, Centaurea cyanus, Centaurium erythraea, Centella asiatica, Cetraria islandica, Chamaemelum nobile, Chamomilla recutita, Chrysanthellum americanum, Cichorium endivia, Cichorium intybus, Cinnamomum zeylanicum, Citrus aurantium, Combretum micranthum, Crataegus oxyacantha, Cuminum cyminum, Cupressus sempervirens, Curcuma zedoaria, Cynara scolymus, Cytisus scoparius, Elettaria cardamomum, Eleutherococcus senticosus, Epilobium parviflorum, Erysimum officinale, Eucalyptus globulus, Eupatorium cannabinum, Foeniculum vulgare, Fraxinus excelsior, Fucus vesiculosus, Fumaria officinalis, Galium odoratum, Gentiana lutea, Geranium robertianum, Ginkgo biloba, Glechoma hederacea, Glycyrrhiza glabra, Handroanthus impetiginosus, Harpagophytum procumbens, Hieracium pilosella, Humulus lupulus, Hypericum perforatum, Hyssopus officinalis, Illicium verum, Inula helenium, Juglans regia, Juniperus communis, Lamium album, Lavandula angustifolia, Levisticum officinale, Lippia citriodora, Lotus corniculatus, Lythrum salicaria, Malva sylvestris, Marrubium vulgare, Medicago sativa, Melissa officinalis, Mentha x piperita, Morus nigra, Myrtus communis, Olea europaea, Origanum majorana, Panax ginseng, Papaver rhoeas, Parietaria officinalis, Passiflora incarnata, Petroselinum crispum, Peumus boldus, Phaseolus vulgaris, Pimpinella anisum, Plantago lanceolata, Plantago ovata, Potentilla anserina, Quercus robur, Rhamnus frangula, Rheum palmatum, Rosa centifolia, Rosmarinus officinalis, Rubia tinctorum, Rubus idaeus, Salix alba, Salvia officinalis, Sambucus nigra, Satureja montana, Silybum marianum, Solanum dulcamara, Tabebuia impetiginosa, Tanacetum vulgare, Taraxacum officinalis, Thymus serpyllum, Thymus vulgaris, Tilia tomentosa, Tilia cordata, Trigonella foenum-graecum, Tussilago farfara, Vaccinium myrtillus, Valeriana officinalis, Verbascum thapsus, Verbena officinalis, Viscum album, Zea mays and Zingiber officinale.

A further object of the invention is an extract of an herbal composition comprising from three to seven dried plants selected among Filipendula ulmaria, Camellia sinensis, Arctostaphylos uva-ursi, Rheum palmatum, Rosmarinus officinalis, Vitis vinifera tinctoria, Desmodium adscendes, Eugenia caryophyllus and Eucalyptus globulus, wherein at least two of such plants are selected among Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi and at least one of such plants is selected among Vitis vinifera tinctoria, Desmodium adscendes and Eugenia caryophyllus.

Another object of the invention is an extract of an herbal composition comprising the following dried plants: Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria and Arctostaphylos uva-ursi.

Another object of the invention is an extract of an herbal composition comprising the following dried plants: Rheum palmatum, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria and Arctostaphylos uva-ursi.

Another object of the invention is an extract of an herbal composition comprising the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria, Eugenia caryophyllus and Arctostaphylos uva-ursi.

A further object of the invention is an extract of an herbal composition comprising the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria, Eucalyptus globulus, Arctostaphylos uva-ursi, Mentha spicata and Rubia tinctorium.

Another object of the invention is an extract of an herbal composition comprising at least fourteen or fifteen dried plants selected among the following sixteen: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera tinctoria, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, Lippia citrodora and Tanacetum vulgare

Another object of the invention is an extract of an herbal composition comprising the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera tinctoria, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum and Lippia citrodora.

Another object of the invention is an extract of an herbal composition comprising the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera tinctoria, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, Lippia citrodora and Tanacetum vulgare.

All the above-mentioned herbal compositions are also an object of the invention, as intermediates in the preparation of the corresponding extracts.

The extract may constitute the active ingredient of a food supplement, a nutraceutical, pharmaceutical or cosmetic composition or a functional food or a food additive.

The extract of the invention may be used as such or in a dried or lyophilized form.

A food supplement, a nutraceutical, pharmaceutical, veterinary or cosmetic composition or a functional food or a food additive containing the extract defined above together with suitable excipients are further objects of the invention.

Such food supplement, a nutraceutical, pharmaceutical, veterinary or cosmetic composition or a functional food or a food additive may be formulated in any form suitable, for example, for oral administration, including solid forms such as powders, granules, capsules, pills, tablets (normal or controlled release), chewing gums, or liquid forms such as syrups, drops, elixirs, solutions and suspensions in general.

Furthermore, the extract according to the invention can be incorporated into other formulations, such as creams, ointments, gels, milks, pastes, creams based on water, emulsions, serums, masks, balms, fluids, sprays, suppositories, vaginal suppositories, transdermal patches and toothpastes, periodontal gels, mouthwashes.

According to the present invention, the expression “herbal composition” refers to a powder containing one or more different grinded dried plants. The powder of a single dried plant may have been obtained from the whole plant or form a specific part of it, for example, flowers, buds, fruits, stems, leaves, seeds, roots or other.

According to the invention, an extract may be obtainable by simple incubation of an herbal composition with an extraction solvent (with or without the addition of a sugar) at room temperature.

Suitable extraction solvents according to the invention are water and all non-aqueous solvents authorized for use in the production of foodstuffs and food ingredients. For example, according to Directive 2009/32/EC a list of such non-aqueous solvents includes propane, butane, ethyl acetate, ethanol, carbon dioxide, acetone, nitrous oxide. According to specific embodiments of the invention, the extraction solvent is selected among water, ethanol, ethyl acetate and any mixture thereof.

According to another alternative embodiment, an extract may be obtainable by incubation of an herbal composition with an extraction solvent (with or without the addition of a sugar) and subsequent heating, with or without centrifugation and filtration.

According to an alternative embodiment, a water extract may be obtainable by incubation of an herbal composition with water (with or without the addition of a sugar) and subsequent heating (for example by heating the resulting liquid preparation at a temperature ranging from 60° C. to 134° C., preferably from 119° to 121° C., for a time ranging from 5 to 60 minutes, preferably from 20 to 30 minutes).

With the term “sugar”, we herein intend to mean a pharmaceutical or alimentary grade sugar, such as for example dextrose, lactose, anhydrous lactose, mannitol, sorbitol, maltose, galactose or sucrose.

According to a particular embodiment of the invention, such sugar is sucrose.

Said sugar may be used at concentrations varying from 1 to 100 g/l, optionally from 50 to 75 g/l.

With the term “food supplement” it is here meant a food product intended to supplement the common diet and which is a concentrated source of nutrients, such as vitamins and minerals, or other substances with a nutritional or physiological effect, in unit dose form (see also Directive 2002/46/EC of 10 Jun. 2002.

The expression “functional food” means to include foods characterized by additional effects due to the presence of components (generally non-nutrients) naturally present or added, which interact more or less selectively with one or more physiological functions of the organism (biomodulation) leading to a positive effect on maintaining health and/or disease prevention. A food can be considered ‘functional’, if it is sufficiently demonstrated its beneficial influence on one or more functions of the body, in addition to adequate nutritional effects, so as to be relevant to a state of well-being and health, or for risk reduction of an illness. The beneficial effects could include both in maintaining that in promoting a state of well-being or health and/or a reduction in the risk of a disease process or disease (see AT Diplock et al: Scientific concepts of functional foods in Europe: Consensus Document, British Journal of Nutrition 1999, 81 (Suppl. 1), S1-S27).

According to the present invention with the expression, “nutraceutical composition” it is intended to mean a product, which not only supplement the diet, but should also aid in the prevention and/or treatment of disease and/or disorder. This expression was coined in 1989 by Stephen L. DeFelice (MD, founder and chairperson of the Foundation for Innovation in Medicine (FIM)) joining the concepts of “nutrition” and “pharmaceutical”.

With the term “excipient”, it is intended here to refer to conventional excipients, i.e., inert compounds towards the active ingredient of a composition.

Excipients may be divided into various functional classifications, depending on the role that they are intended to play in the resultant formulation, e.g., in solid dosage forms they may act as diluents (e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycolate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal SiO2) or flavoring agents (e.g. peppermint, lemon oils). In liquid dosage forms they may act as solvents, co-solvents or solubilizers (e.g., propylene glycol, glycerol, ethanol, sorbitol, dextrose), buffering agents (e.g. sodium phosphate), antimicrobial agents (e.g. potassium sorbate), wetting agents (e.g. polysorbates or Tweens), anti-foaming agents (e.g. polydimethylsiloxane-silicon dioxide), thickening agents (e.g. methylcellulose or hydroxyethylcellulose) or sweetening agents (e.g. sorbitol, acesulfame-K).

As already said, the extract of the invention is an antimicrobial and/or antibiofilm agent and it is advantageously used in the treatment, the prevention and/or the diagnosis of microbial infections in humans and animals.

Another object of the invention is therefore the use of the extract of the invention in the manufacture of a medicament for the treatment, prevention or diagnosis of microbial infections. Such medicament may be useful for humans of animals.

According to the present invention, the expressions “treatment” or “treating” are intended to activities designed to cure, mitigate the symptoms or delay the progression of a disease or a pathological condition.

The expressions “prevention” or “preventing” are intended to refer to activities designed to minimize the incidence or effects of a disease or a pathological condition. For example, in the case of microbial infections, prevention specifically herein means to prevent the adhesion and colonization of pathogenic microbes to cells, tissues and organs.

The expressions “diagnosis” or “diagnosing” are intended to refer to activities designed to identify a series of physiological parameters in order to assess the presence and the extent of a determined disease or pathological condition.

The expression “antimicrobial agent” will here be used to mean an agent that is able to inhibit the growth of microbial cells. If the microbial cells are pathogenic microbial cells, the antimicrobial agent can be used as active ingredient of a pharmaceutical or veterinary composition for the treatment of microbial infections in humans or animals.

The expression “antibiofilm agent” will here be used to mean an agent that is able to inhibit the formation of biofilm by microbial cells and/or to facilitate the dispersion of preformed biofilms. In contrast to conventional antimicrobial agents, an antibiofilm agent does not directly affect microbial survival and thus the expectation is that resistance to these agents will not readily occur.

According to the present invention, the term “synergistic” is intended as referred to a biological effect or activity (herein antimicrobial or antibiofilm), presented by the extract of an herbal composition of the invention (i.e. comprising at least two dried plants selected among Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi), which is greater than the sum of the effects or activities of the extracts of the corresponding single dried plants.

Such greater biological effect or activity may be illustrated, for example, in terms of inhibition of planktonic growth (antimicrobial activity), in terms of biofilm formation inhibition (antibiofilm activity) or in terms of number of strains, in which one or both these activities can be identified.

According to a particular embodiment of the invention, the microbial infections are caused by bacteria, such as for example Escherichia coli, Klebsiella pneumoniae, Listeria innocua, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica subsp. Enterica serovar Enteritidis, Salmonella enterica subsp. Enterica serovar Typhimurium, Streptococcus pneumoniae, Streptococcus equi subsp. zooepidemicus, Streptococcus pyogenes, or by fungi, such as for example Candida albicans.

According to another particular embodiment of the invention, the microbial infections are caused by Staphylococcus aureus, Staphylococcus epidermidis or Staphylococcus pseudintermedius.

Staphylococcus aureus, a commensal Gram-positive bacterium is a leading pathogen that may cause a wide variety of diseases in human ranging from skin and soft tissue infections to severe septicemia, toxic choc or pneumonia. Staphylococcus aureus is an important cause of hospital-associated infections, and many hospital strains of Staphylococcus aureus have acquired resistant to antibiotics, most particularly to methicillin. There is no vaccine available yet, but important efforts are conducted to develop active and passive immunotherapy against various targets of Staphylococcus aureus.

In order to evaluate such biological activities for the extract of the invention and for comparative samples, many different strains have been used and will be mentioned in the Examples' section.

In particular, the following bacterial strains have been used to test both the antimicrobial and antibiofilm activity of the extracts of the invention.

ATCC 25923, also known as Seatle 1945, is a Staphylococcus aureus strain widely used in academic research (it is cited in 627 publications as of October 2016 (see NIH-NCBI-Pubmed)). Its antibiotic resistance behavior has been thoroughly characterized (see, for instance, Reimer L. G. et al., Antimicrob. Agents Chemother. 1981 June; 19(6):1050-5).

ATCC 29213 is also a Staphylococcus aureus strain widely used in academic research (cited in 327 publications as of October 2016 (see NIH-NCBI-Pubmed)). It has been used in biofilm research (see, for instance, Ceri H. et al., J. Clin. Microbiol. 1999 June; 37(6):1771-6).

ATCC 1228 is a Staphylococcus epidermidis strain (cited in 101 publications as of October 2016 (see NIH-NCBI-Pubmed)). The Code of Federal Regulations (FDA) lists such strain as the preferred assay organism for neomycin (see also Robertson J. H. et al., Appl. Microbiol. 1971 December; 22(6):1164-5).

ATCC 43300 is a Staphylococcus aureus strain isolated from a clinical study (cited in 81 publications as of October 2016 (see NIH-NCBI-Pubmed), including Lubenko et al., J. Antimicrob. Chemother. 2008 November; 62(5):1065-9).

Staphylococcus aureus strain Newman was isolated in 1952 from a human infection and has been used extensively in animal models of staphylococcal infections due to its robust virulence phenotype (see Baba T. et al., Journal of bacteriology, 190:300-310 (2008)). In contrast to hospital-acquired MRSA (Methicillin-resistant Staphylococcus aureus), Staphylococcus aureus Newman carries a harbors only small a small number of insertion sequences (IS) and lacks known antibiotic resistance determinants.

Other strains used in the examples are also commonly used in academic research or in industry for quality control (QC). Most of them are also methicillin resistant.

For reference to ATCC 33591, see Reyes N. et al., J. Antimicrob. Chemother. 2006 August; 58(2):462-5, Epub 2006 May 30. For reference to ATCC 33592, see Teka A. et al., BMC Complement Altern Med. 2015 Aug. 18; 15:286. For reference to NCTC 12493, see Carey B. E. et al., J. Antimicrob. Chemother. 2006 August; 58(2):455-7, Epub 2006 Jun. 20. For reference to ATCC 700698, see Kirker K. R. et al., Int. J.

Antimicrob Agents, 2015 October; 46(4):451-5, Epub 2015 Jul. 9. For reference to ATCC 700699, see Sy C. L. et al., J. Clin. Microbiol. 2016 March; 54(3):565-8, Epub 2015 Dec. 16. For reference to ATCC 9144, see Carson C. F. et al., Antimicrob. Agents Chemother. 2002 June; 46(6):1914-20. For reference to ATCC BAA-44, see Monecke S. et al., PLoS One. 2011 Apr. 6; 6(4): e17936. For reference to ATCC 14990, see Bernardo T. H. et al., Scientific World Journal. 2015; 751791, Epub 2015 Jun. 3). For reference to ATCC 49444, see Ramsey K J et al., J Food Prot. (2010). For reference to ATCC 60193, see Petrikaite V et al., Medicina (Kaunas). 2007; 43(8):657-63. For reference to ATCC 11775, see Usman H et al., Afr J Tradit Complement Altern Med. 2007 Jun. 10; 4(4):488-94. For reference to ATCC 13883, see Supardy N A et al., J Microbiol Biotechnol. 2012 June; 22(6):872-81. For reference to ATCC 33090, see Le Marc Y et al., Int J Food Microbiol. 2002 March; 73(2-3):219-37. For reference to ATCC 19115, see Vodnar D C., Chem Cent J. 2012 Jul. 28; 6(1):74. doi: 10.1186/1752-153X-6-74. For reference to ATCC 27853, see Reimer L G et al., Antimicrob Agents Chemother. 1981 June; 19(6):1050-5. For reference to ATCC 13076, see Fioretto F et al., Braz J Med Biol Res. 2005 August; 38(8):1259-65. Epub 2005 Jul. 30. For reference to ATCC 13311, see Leguérinel I et al., Int J Food Microbiol. 2007 May 1; 116(1):88-95. Epub 2007 Jan. 13. For reference to ATCC 27336, see Chen S et al., Appl Environ Microbiol. 2013 July; 79(13):4015-23. doi: 10.1128/AEM.00704-13. Epub 2013 Apr. 19. For reference to ATCC 43079, see Farrow J A and Collins M D., Syst. Appl. Microbiol. 5: 483-493, 1984. For reference to ATCC BAA-1323, see Svensson Mikael D et al., Microbiology 148: 3933-3945, 2002.

The food supplement, the nutraceutical, pharmaceutical or cosmetic composition of the invention may be suitably formulated for different routes of administration. Such routes of administrations include auricular, buccal, cutaneous, dental, nasal, ophthalmic, oral, oropharyngeal, rectal, respiratory (inhalation), sublingual, topical or vaginal.

Generally, the compositions comprising the extracts of the invention are administered in a “therapeutically effective amount”.

For the aim of the present description and of the following claims, the term “comprising” also includes the terms “which essentially consists of” or “which consists of”.

The amount of the composition actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual composition administered, the age, body weight, and response of the individual subject, the severity of the subject's symptoms and the like. For any composition, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rats, guinea pigs, rabbits, dogs, or pigs.

The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. In calculating the Human Equivalent Dose (HED) it is recommended to use the conversion table provided in Guidance for Industry and Reviewers document (2005, U.S. Food and Drug Administration, Rockville, Md., USA).

According to the invention, the dose of the extract to be administered to humans may be regulated referring to the herbal composition used to prepare it and, consequently, the amount of grinded single dried plants present in the herbal composition. Usually, from 0.001 to 500 g of each grinded dried plant is used to prepare the final extract, optionally from 0.01 to 100 g. Preferably, from 1 to 100 g of each grinded dried plant is used per 1 kg of final extract. Such extract may be used as such or in a lyophilized, dried or freeze dried form in the final composition for human or animal use or it may be further diluted.

Preferably, from 0.001 to 5 g of each grinded dried plant is used per 1 kg of final product on sale.

The precise effective dose for a human subject will depend upon the severity of the disease or condition, general health of the subject, age, weight and gender of the subject, diet, time and frequency of administration, other simultaneous therapies, reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician.

Dosage treatment may be a single dose schedule or a multiple dose schedule.

Another object of the invention is the process for preparing the extract of the invention. Such process schematically comprises the following steps:

    • a) chopping or grinding separately or in a mixture at least two single dried plants to obtain corresponding plant powders;
    • b) mixing at least two different plant powders to obtain an herbal composition;
    • c) adding an extraction solvent to said herbal composition to obtain a corresponding liquid preparation (or extract);
    • d) incubating said liquid preparation at room temperature for a time ranging between 5 and 15 minutes; optionally,
    • e) heating said liquid preparation at a temperature ranging from 60° C. to 134° C. for a time ranging from 5 to 60 minutes; optionally,
    • f) centrifuging, collecting the supernatant and filtering the collected supernatant; and, optionally,
    • g) concentrating and/or drying or freeze drying the liquid preparation obtained from step e) or f).

According to the present application the word “lyophilisate” will refer to both freeze-dried (de-dehydrated) and dried extracts according to the invention.

The single dried plants are commercial products that may be purchased as part of the dried plants collections of pharmacies or herbal shops. Lists of dried plants collections are available at each pharmacy of herbal shop of choice and the dried plants available in such collections may vary from country to country.

For example, Belgium, France and Italy, decided in 2012 to join forces to develop a common list of plants, whose use could be allowed, provided that manufacturers meet the quality requirements of European law (EC Regulation 852/2004). Thanks to the hard work of three recognized experts (Robert Anton, Luke Delmulle and Mauro Serafini) the project resulted in the establishment of a list of 1,029 plants and 11 mushrooms (see list BelFrit at

http://www.economie.gouv.fr/fils/fils/directions_services/dgccrf/imgs/breve/2014/documents/ha rmonized_list_Section_A.pdf)

The invention will now be described by making reference to the following non-limiting Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the growth kinetics of Pseudomonas aeruginosa ATCC®27853™ at 37° C. in TS broth, OD measured in 96 well plate, with the following final dilution of processed sample: 1:10.

FIG. 2 shows the close-up growth kinetics of Pseudomonas aeruginosa ATCC® 27853™ at 37° C. in TS broth, OD measured in 96 well plate, with the following final dilution of processed sample: 1:10.

FIG. 3 shows the growth kinetics of Staphylococcu aureus subsp. aureus ATCC® 29213 at 37° C. in TS broth, OD measured in 96 well plate, with the following final dilution of processed sample: 1:40.

FIG. 4 shows the close-up growth kinetics of Staphylococcu aureus subsp. aureus ATCC® 29213 at 37° C. in TS broth, OD measured in 96 well plate, with the following final dilution of processed sample: 1:40.

 EXAMPLES List of Plants Used in the Examples

The plants and parts of plants in the following list have been used in the different Examples. For the sake of conciseness, each plant is identified by a code used throughout the Examples. The weight corresponding to 200 μl (volume of the calibrated spoon) of plant powder is also indicated (Table 0-1). The dried plants of pharmaceutical grade PA00 to PB12 were obtained from “Pharmacie Fontgiève” (Clermont-Ferrand, France) and the dried plants of pharmaceutical grade PB13 to PD08 were obtained from “Pharmacie St Herem” (Clermont-Ferrand, France). When indicated in the Example, plants were also obtained from “Herboristerie Cailleau” (Chemillé. France) or from Siccarapam (Aubiat, France).

TABLE 0-1 List of plants with associated codes and weights of dried powders for one calibrated spoon Plant Weight Code Botanic Name Part used (g) PA00 Sambucus nigra flower 0.0684 PA01 Plantaga afra seed 0.1357 PA02 Eguisetum arvense aerial part 0.0578 PA05 Aloysio citrodora leaf 0.0712 PA06 Glycyrrhiza glabra root 0.0774 PA07 Camellia sinensis leaf 0.07 PA08 Solidago virg aurea aerial part 0.0394 PA10 Taraxacum officinale root 0.0585 PA11 Rheum palmatum rootand 0.1016 rhizome PA12 Rosmarinus leaf 0.0545 officinalis PA13 Filipendula ulmaria flower 0.095 PA14 Satureja montana leaf 0.0619 PA15 Calendula officinalis petal 0.0383 PA16 Salvia officinalis leaf 0.0415 PA17 Passiflora incarnata aerial part 0.0423 PA18 Plantago lanceolata leaf 0.0626 PA19 Tussilago farfara inflorescence 0.0494 PA20 Valeriana officinalis root 0.1142 PA21 Camellia sinensis leaf 0.135 PA22 Vitis vinifera var. leaf 0.0762 tinctoria PA23 Thymus vulgaris leaf 0.0373 PA24 Cassia angustifolia leaflet 0.0648 PA25 Tilia cordata sapwood 0.0391 PA26 Thymus serpyllum aerial part 0.0442 PA27 Tilia tomentosa bract 0.0775 PA28 Rubus idaeus leaf 0.0463 PA29 Fucus vesiculosus lamina 0.1702 PA30 Lavandula flower 0.0216 angustifolia PA31 Trigonella foenum- seed 0.1043 graecum PA32 Lotus corniculatus aerial part 0.0497 PA33 Handroanthus bark 0.0814 impetiginosus PA34 Viscum album leaf 0.0783 PA35 Fumaria officinalis flowering top 0.0661 PA36 Humulus lupulus fruit 0.0307 PA37 Fraxinus excelsior leaf 0.0647 PA38 Cytisus scoparius flower 0.0253 PA39 Foeniculum vulgare fruit 0.081 PA40 Origanum majorana leaf 0.0345 PA41 Citrus aurantium var. leaf 0.0574 dulcis PA44 Hyssopus officinalis flowering top 0.0677 PA45 Mentha x piperita leaf 0.07 PA46 Melissa officinalis leaf 0.0747 PA47 Fragaria vesca whole plant 0.0692 PA48 Phaseolus vulgaris fruit 0.0608 PA51 Marrubium vulgare aerial part 0.0457 PA52 Malva sylvestris flower 0.0355 PA53 Citrus aurantium var. petal 0.0851 dulcis PA55 Ginkgo biloba leaf 0.0701 PA57 Lamium album flower 0.0591 PA60 Olea europaea leaf 0.0572 PA61 Ilex paraguariensis leaf 0.0598 PA62 Artemisia vulgaris leaf 0.0447 PA67 Epilobium aerial part 0.0549 parviflorum PA69 Chamomilla recutita inflorescence 0.0399 PA70 Sisymbrium aerial part 0.0334 officinale PA72 Asparagus officinalis root 0.0523 PA73 Pimpinella anisum fruit 0.0993 PA74 Ribes nigrum fruit 0.0769 PA75 Crataegus flowering top 0.0656 oxyacantha PA76 Chamaemelum inflorescence 0.0193 nobile PA79 Eucalyptus globulus leaf 0.0703 PA80 Prunus cerasus peduncle 0.0141 PA81 Acorus calamus rhizome 0.0631 PA82 Calluna vulgaris flower 0.0493 PA83 Papaver rhoeas petal 0.0278 PA85 Cumimum cymimum fruit 0.102 PA86 Centaurium aerial part 0.0591 erythraea PA88 Vaccinium myrtillus fruit 0.0939 PA89 Vaccinium myrtillus leaf 0.0711 PA90 Angelica fruit 0.0388 archangelica PA92 Borago officinalis flower 0.067 PA93 Alchemilla vulgaris aerial part 0.0588 PA94 Artemisia pontica flowering top 0.431 PA95 Illicium verum fruit 0.0793 PA96 Chrysanthellum aerial part 0.0768 americanum PA97 Harpagophytum root 0.0853 procumbens PA98 Betula pendula leaf 0.0633 PA99 Plantago lanceolata aerial part 0.0718 PB00 Agropyrum repens rhizome 0.0646 PB01 Carum carvi fruit 0.1089 PB02 Buxus sempervirens leaf 0.0631 PB03 Cichorium endivia root 0.1513 PB04 Rosa canina fruit 0.1227 PB05 Silybum marianum aerial part 0.0502 PB06 Asperula odorata aerial part 0.062 PB07 Desmodium leaf 0.0493 adscendens PB08 Combretum leaf 0.0709 micranthum PB09 Peumus boldus foliole 0.0574 PB10 Rhamnusfrangula bark 0.0682 PB11 Arctium lappa root 0.1166 PB12 Arctostaphylos leaf 0.122 uva-ursi PB13 Cichorium intybus root 0.0924 PB14 Fragaria vesca rhizome 0.0529 PB15 Citrus aurantium var. peel 0.0817 dulcis. PB16 Elettaria fruit 0.0606 cardamomum PB17 Althaea officinalis leaf 0.0302 PB18 Malva sylvestris leaf 0.0809 PB19 Althaea officinalis leaf 0.0322 PB20 Tilia cordata flowering top 0.0596 PB22 Betula pendula leaf 0.0609 PB24 Tilia cordata sapwood 0.0368 PB25 Fucus vesiculosus lamina 0.1119 PB26 Borago officinalis flower 0.0342 PB27 Vaccinium myrtillus fruit 0.1553 PB28 Verbascum thapsus flower 0.045 PB29 Citrus aurantium petal 0.0477 PB31 Galega officinalis aerial part 0.0678 PB32 Tabebuia bark 0.0775 impetiginosa PB33 Giechoma aerial part 0.0601 hederacea PB35 Cetraria islandica aerial part 0.0776 PB36 Rubus idaeus leaf 0.0553 PB38 Fraxinus excelsior leaf 0.1241 PB39 Lavandula flower 0.0207 angustifolia PB41 Cassia angustifolia fruit 0.0568 PB42 Fragaria vesca leaf 0.0692 PB45 Aspalathus linearis leaf 0.0684 PB46 Trigonella foenum- seed 0.1342 graecum PB47 Foeniculum vulgare fruit 0.0818 PB48 Alkanna tinctoria root 0.0606 PB49 Mentha spicata leaf 0.0694 PB50 Eupatorium aerial part 0.0529 cannabinum PB51 Echinacea purpurea root 0.0915 PB55 Eucalyptus globulus leaf 0.0666 PB57 Rosaxcentifolia bud 0.0819 PB58 Solanum dulcamara stem 0.0709 PB60 Arbutus unedo leaf 0.0891 PB61 Borrago officinalis aerial part 0.0754 PB76 Eleutherococcus root 0.0571 senticosus PB77 Cupressus nut 0.0862 sempervirens PB78 Curcuma longa root 0.0946 PB79 Cuminum cyminum fruit 0.1109 PB81 Salvia officinalis leaf 0.0536 PB82 Salix alba bark 0.0522 PB83 Saponaria officinalis aerial part 0.0602 PB85 Lythrum salicaria aerial part 0.0581 PB86 Citrus limon (L) peel 0.098 Bumf. PB88 Centella asiatica aerial part 0.0685 PB92 Plantago ovata seed 0.1266 PB97 Agropyrum repens rhizome 0.0555 PB98 Cichorium intybus leaf 0.0715 PB99 Potentilla anserina leaf 0.0298 PC01 Ouercus robur bark 0.06 PC02 Chelidonium majus aerial part 0.0608 PC04 Prunus cerasus peduncle 0.0199 PC06 Asparagus officinalis root 0.0812 PC08 Medicago sativa seed 0.113 PC11 Juglans regia leaf 0.0641 PC12 Olea europaea leaf 0.0561 PC14 Myrtus communis leaf 0.0716 PC15 Trifolium rubens flower 0.0484 PC17 Curcuma zedoaria rhizome 0.077 PC19 Vitis vinifera var. leaf 0.0695 tinctoria PC20 Eugenia clove 0.0852 caryophyllus PC21 Althaea officinalis root 0.0642 PC22 Alpinia officinarum rhizome 0.0658 PC23 Ginkgo biloba leaf 0.0775 PC24 Panax ginseng lateral root 0.1782 PC25 Zingiber officinale rhizome 0.078 PC26 Juniperus communis fruit 0.0505 PC27 Geranium aerial part 0.0639 robertianum PC28 Gentiana lutea root 0.0992 PC29 Rubia tinctorium root 0.0868 PC32 Jasminum officinale flower 0.0616 PC33 Combretum leaf 0.0769 micranthum PC34 Satureja montana leaf 0.0715 PC35 Cassia angustifolia leaflet 0.0881 PC36 Verbena officinalis aerial part 0.0603 PC37 Lippia citrodora leaf 0.0813 PC38 Veronica officinalis complete 0.0781 PC41 Calendula officinalis petal 0.0523 PC42 Sambucus nigra bark 0.0593 PC45 Solidago gigantea aerial part 0.0612 PC49 Tanacetum vulgare aerial part 0.0577 PC50 Galium odoratum aerial part 0.041 PC51 Rhamnusfrangula bark 0.0726 PC52 Betula alba bark 0.0806 PC53 Verbascum thapsus bark 0.0435 PC54 Peumus boldus leaf 0.0695 PC55 Centaurea cyanus petal 0.0456 PC57 lllicum verum fruit 0.1027 PC58 Inula helenium root 0.0904 PC60 Cinnamomum bark 0.1374 zeylanicum PC61 Chamaemelum flower 0.0316 nobile PC62 Calamintha aerial part 0.0726 officinalis PC63 Arctostaphylosu vaursi leaf 0.088 PC64 Ononis spinosa root 0.0832 PC65 Calluna vulgaris flower 0.0446 PC66 Morus nigra leaf 0.1032 PC67 Hypericum flowering top 0.0829 perforatum PC68 Achillea millefolium flowering top 0.0332 PC70 Mentha × piperita leaf 0.071 PC71 Melissa officinalis leaf 0.0847 PC72 Cochlearia officinalis flowers 0.0497 PC73 Malva sylvestris flower 0.0615 PC74 Cynara scolymus leaf 0.0458 PC75 Arnica montana flower 0.0254 PC76 Artemisia vulgaris aerial part 0.0289 PC78 Angelica complete 0.0909 archangelica PC79 Anethum graveolens fruit 0.1335 PC82 Ballota nigra aerial part 0.0368 PC83 Origanum majorana leaf 0.0539 PC84 Zea mays stigma 0.0632 PC86 Agrimonia eupatoria aerial part 0.0721 PC88 Levisticum officinale aerial part 0.0848 PC94 Phaseolus vulgaris fruit 0.0686 PC97 Hieracium pilosella aerial part 0.0406 PC98 Parietaria officinalis aerial part 0.0349 PC99 Passiflora incarnata aerial part 0.0817 PD00 Rumex patientia root 0.1208 PD02 Petroseiinum fruit 0.1084 crispum PD05 Urtica dioica aerial part 0.0584 PD06 Cola acuminata nut 0.1556 PD07 Capsellabursapastoris aerial part 0.0535 PD08 Centaurea erythraea aerial part 0.058

Method for Preparing the Herbal Compositions (Method A)

Each dried plant was grinded using a coffee bean grinder (Type 8100, SEB, France). The obtained powder (plant powder) was sieved using a 2 mm mesh sieve, and conserved in 60 ml containers (Labbox, France).

Mixtures of different grinded dried plants (or plant powders) were prepared by collecting a fixed volume of plant powder with a calibrated spoon allowing to collect 200 μL of powder. The weight of the collected volume is dependent on the specific plant powder (see Table 0-1).

Such mixes or mixtures of different grinded dried plants (or plant powders) as well as single dried plants will herein be called also as “herbal compositions”.

Method for Preparing the Water Extracts (Also Called in this Examples Section as “Processed Samples”, “Extracts” or “Mixes Extracts”) (Method B)

Such herbal compositions were placed into 50 ml falcon tubes (Falcon, USA) and 20 ml or 40 ml (as indicated in Examples) of alimentary grade spring water (Volvic, France) solution containing 100 g/l of alimentary grade sucrose (Daddy, France) were added. The choice of alimentary grade sucrose and water rather than their scientific grade counterparts was dictated by the objective of obtaining an edible composition entering the class of “flavourings” that may be directly consumed under the Regulation (EC) No 1334/2008 of 16 Dec. 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods.

Each falcon tube containing such liquid preparations was agitated using a vortex mixer (ThermoFisher, USA) for 10 seconds, incubated at room temperature for 10 min and placed in an autoclave (MED 12, JP Selecta, Spain) and underwent a 20 minute sterilization cycle at 119° or 121° C., as indicated. After cooling down, the sterilized preparation was kept at −20° C. until use.

The day of manipulation, the preparation was defrosted at 37° C. The preparation was centrifuged at 4000 rpm during 10 min (Multifuge 35-R, Heraeus, Germany) or incubated at room temperature for decantation (as indicated in Examples). 2 mL of the supernatant was collected with a 10 mL syringe and filtered using 0.2 μM filters (Minisart Syringe Filters, Sartorius, Germany) and collected onto a 2 mL Eppendorf tube. The remaining sterilized preparation is kept at −20° C. for further use.

The samples so obtained will herein be called also as “processed samples” and will be identified by the same name or code given to the corresponding herbal composition (for example the name “Mix[2P]-1” will be used to identify an herbal composition and the corresponding extract (or processed sample), for which a biological activity is determined).

Method for Determining the Antimicrobial and Antibiofilm Activity (Method C)

The biological activity of each processed sample was determined by using the strains indicated in each experiment (or Example). For each measurement, each bacterium stored at −80° C. on cryobeads (Technical Service, UK) is plated on a TS agar plate (Tryptic Soy Broth, Sigma, USA; Agar, Fisherbrand, USA) and incubated at 37° C. overnight. Several colonies are collected using an inoculation loop and suspended in saline solution. The optical density is measured, and a dilution is performed in TS broth to obtain a concentration of 2.105.5 bacteria/mL for Staphylococcus aureus and Staphylococcus pseudintermedius strains, 2.105 bacteria/mL for Staphylococcus epidermidis.

The biological activity of each processed sample was measured on 96 wells plates, using the strains indicated in each experiment (or Example) and a control without bacteria. Each well contains 190 μL of TS broth inoculated with 105.5 bacteria/mL for Staphylococcus aureus and Staphylococcus pseudintermedius strains, 105 bacteria/mL for Staphylococcus epidermidis (or at the concentration indicated in the Examples for all other species) or no bacteria for the control and diluted processed sample or negative control (sucrose solution at 100 g/l without any plants) at 1:10, 1:20, 1:40 and 1:80 final concentration or at 1:20, 1:63 and 1:200 (as indicated in Examples).

Each plate was incubated for 17 hours at 37° C. in an incubator (HettCube 400, Hettich, Germany).

The measurements were performed based on analyzing with the ImageJ software (NIH, USA) (but any other equivalent software may be used) images of the plates obtained using a commercial document scanner (V 220, Epson, Japan). Images of the plate positioned on the scanning area are acquired at 600 dpi using the software provided with the scanner. For each plate, 3 images were acquired: the first, before incubation (T0), the second after 17 h incubation (Tf), and the third after Crystal Violet (CV) staining as described in Optimization of microtitre plate assay for the testing of biofilm formation ability in different Salmonella serotypes (Agarwal, R. K. et al., International Food Research Journal 18(4):1493-1498, 2011).

For each well of interest, from respectively the first, second and the third image, circular regions of diameter, respectively, 14, 14 and 76 pixels, centered relative to the well, were characterized using an ImageJ macro to calculate, respectively, the mean M1 and M2 of the 1% less intense pixels of the region and the mean M3 of all the pixels of the region. The turbidity of the bacterial solution in the well was deduced from M1 and M2 using a reference curve obtained in an independent experiment where the mean of the 1% less intense pixels of the region 14 pixels diameter region was measured from wells containing bacterial solutions of known turbidity. The optical density of the Crystal Violet solution in the well was deduced from M3 using a reference curve obtained in an independent experiment where the mean of the pixels of the region 76 pixels diameter region was measured from wells containing Crystal Violet solutions of known optical density.

For each processed sample and a given bacterium, the MIC was determined from the highest inhibitory dilution (HID) among the 4 tested (1:10, 1:20, 1:40, 1:80) of processed sample which prevents the appearance of visible growth within 17 h. “0” was assigned when none of the tested dilutions was preventing bacterial growth.

For each well containing processed sample and a given bacterium, the inhibition percentage of bacteria planktonic growth was established according to the formula below:

Planktonic ⁢ growth ⁢ inhibition ⁢ ( % ) = ( 1 - Tf ⁡ ( processed ⁢ sample ) - T ⁢ 0 ⁢ ( processed ⁢ sample ) Tf ⁡ ( negative ⁢ control ) - T ⁢ 0 ⁢ ( negative ⁢ control ) ) * 100

where Tf(processed sample) is the turbidity from the second image of the sample well, T0(processed sample) is the turbidity of the first image of the same well, Tf(negative control) is the turbidity from the second image of a well containing the same bacteria without processed sample, T0(negative control) is the turbidity of the first image of the same well.

For each well containing processed sample and a given bacterium, the percentage of inhibition of biofilm formation (IBF) was established according to the following formula:

Biofilm ⁢ formation ⁢ inhibition ⁢ ( % ) = ( 1 - CV ⁡ ( processed ⁢ sample ) - CV ⁡ ( processed ⁢ sample ) CV ⁡ ( negative ⁢ control ) - CV ⁡ ( negative ⁢ control ) ) * 100

where CV(processed sample) is the OD from the third image of the sample well, CV(negative control) is the OD from the third image of a well containing the same bacteria without processed sample, CV(control sample) is the OD of the third image of a well containing the same sample or the negative control at the same dilution without any bacteria. A negative value of IBF means that the tested mixture is promoting the formation of biofilm.

Example 1

In this Example we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus of the disclosed composition of 2 plants among 3: Filipendula ulmaria, Camellia sinensis, Arctostaphylos uva-ursi. The dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France).

Starting from such three different plant powders six herbal compositions were prepared according to the Method A reported before, whose content is reported in Table 1-1.

TABLE 1-1 herbal compositions of processed samples Herbal PA13 PA21 PB12 compositions (nb spoon) (nb spoon) (nb spoon) PA13 1 PA21 1 PB12 1 Mix[2P]-1 1 1 Mix[2P]-2 1 1 Mix[2P]-3 1 1

As shown in Table 1-1 the first three herbal compositions only contain a single plant powder, while the last three compositions contain two of the three different plant powders.

Starting from each herbal composition reported in Table 1-1 corresponding processed samples were prepared according to the Method B reported before using 20 mL of water containing 100 g/l of sucrose for water extraction and including the centrifugation step before filtration.

The bacteria planktonic growth and antibiofilm formation inhibition percentage of the processed samples of the 2 plant mixes and the associated separated plants of this Example 1 at 1:10 dilution was determined by using the following Staphylococcus aureus strains: ATCC 25923 and ATCC 29213, according to the Method C reported before and are given in Table 1-2.

TABLE 1-2 planktonic growth and biofilm formation inhibition (IBF) of 2 plants mixture processed sample and single plants processed sample at 1:10 dilution PA13 PA21 Mix[2P]-1 ATCC Planktonic growth inhibition 20% 26% 82% 25923 Biofilm formation inhibition 32% 44% 78% ATCC Planktonic growth inhibition 16% 27% 100% 29213 Biofilm formation inhibition 33% 17% 85% PA21 PB12 Mix[2P]-2 ATCC Planktonic growth inhibition 26% 17% 104% 25923 Biofilm formation inhibition 44% 51% 88% ATCC Planktonic growth inhibition 27% 8% 100% 29213 Biofilm formation inhibition 17% 28% 83% PA13 PB12 Mix[2P]-3 ATCC Planktonic growth inhibition 20% 17% 82% 25923 Biofilm formation inhibition 32% 51% 93% ATCC Planktonic growth inhibition 16% 8% 66% 29213 Biofilm formation inhibition 33% 28% 78%

PA13, PA21 and PB12 have a known antibiotic effect. Limited inhibition of planktonic growth (<27%) and biofilm formation (<51%) were measured at 1:10 dilution, whereas the 3 aromatic preparations of 2 plant mix extract show antimicrobial effects (>66%) and antibiofilm effects (>78%) on both tested strains, ATCC 25923 and ATCC 29213.

For all Mixes (Mix[2P]-1, Mix[2P]-2, Mix[2P]-3), a synergistic effect of the 2 plants is observed for antimicrobial effects on the 2 strains tested.

Comparative Example 1

As a comparative Example, we illustrate the activity of some processed samples, which have been found to have none or poor antimicrobial and antibiofilm activities against Staphylococcus aureus and which were obtained from corresponding herbal compositions containing 2 different plant powders.

Five processed samples were prepared using 2 additional plants powders: PA74 (Ribes nigrum fruit powder) and PC72 (Cochlearia officinalis flowers powder).

The single dried plant powders were treated as described in Example 1 and five herbal compositions were prepared following the protocol reported in Method A described before. Such five herbal compositions had the content reported in Table 1-3.

TABLE 1-3 herbal compositions of processed samples Herbal PA74 PC72 PA13 PA21 Compositions (nb spoon) (nb spoon) (nb spoon) (nb spoon) PA74 1 PC72 1 Mix 1 1 1 Mix 2 1 1 Mix 3 1 1

Each herbal composition was treated following the protocol reported in Method B to arrive at the corresponding processed sample using 20 mL of water containing 100 g/l of sucrose for water extraction and including the centrifugation step before filtration.

The biological activity of each processed sample was determined by using the following Staphylococcus aureus strains: ATCC 25923 and ATCC 29213, according to Method C described before.

The bacteria planktonic growth and antibiofilm formation inhibition percentage of the processed samples of the 2 plant mixes and the associated separated plants at 1:10 dilution are given in Table 1-4.

TABLE 1-4 planktonic growth and biofilm formation inhibition (IBF) of 2 plants mixture processed sample and single plants processed sample at 1:10 dilution PC72 PA74 Mix 1 ATCC 25923 Planktonic growth inhibition  −9%   0% −3% Biofilm formation inhibition  12%  36% 24% ATCC 29213 Planktonic growth inhibition −10%  −7% −6% Biofilm formation inhibition  13%   5% −2% PA13 PA74 Mix 2 ATCC 25923 Planktonic growth inhibition  20%   0% 19% Biofilm formation inhibition  32%  36% 48% ATCC 29213 Planktonic growth inhibition  16%  −7% 13% Biofilm formation inhibition  33%   5% 37% PA21 PC72 Mix 3 ATCC 25923 Planktonic growth inhibition  26%  −9% 25% Biofilm formation inhibition  44%  12% 51% ATCC 29213 Planktonic growth inhibition  27% −10% 18% Biofilm formation inhibition  17%  13% 29%

PC72 and PA74 and the associated Mix 1 showed no antimicrobial activity and limited antibiofilm activity on both tested strains at 1:10 dilution. In each case, no synergistic effect was observed.

For all mixes containing PA13 and PA21 (Mix 2 and 3), no synergistic effect was observed for antimicrobial effects on the 2 strains tested at 1:10 dilution, when such mixes contained also PC72 or PA74.

It is interesting to note that both Ribes nigrum and Cochlearia officinalis were known to have antimicrobial effect.

This comparative Example illustrates that not any random combination between single dried plants having known antimicrobial and/or antibiofilm activity allows to obtain a water extract, in which a synergistic activity is observed.

Example 2

In this Example we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus of different water extracts obtained from the herbal compositions containing 2 plants among the 3 reported in Example 1, when such water extracts are prepared according to different methods.

The dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France).

Twenty herbal compositions, whose content is reported in Table 2-1 were prepared according to the Method A reported before.

TABLE 2-1 herbal compositions of processed samples Herbal PA13 PA21 PB12 compositions (nb spoon) (nb spoon) (nb spoon) Mix 1 1 1 Mix 2 1 1

For each herbal composition, 20 liquid (water) preparations were prepared as follow:

The collected powders were placed into twenty 50 ml falcon tubes (Falcon, USA). 20 ml of alimentary grade spring water (Volvic, France) solution containing 0, 25, 50, 75 or 100 g/l of alimentary grade sucrose (Daddy, France) were added to 4 flacon tubes per condition. Each falcon tube containing such liquid preparations was agitated using a vortex mixer (ThermoFisher, USA) for 10 seconds, incubated at room temperature for 10 minutes, then 4 different heating treatments were applied to each condition:

    • Cold Infusion (No Heating Before Centrifugation and Filtration):
    • after incubation at room temperature, the preparations were centrifuged at 4,000 rpm during 10 minutes (Multifuge 35-R, Heraeus, Germany), 2 mL of the supernatant was collected with a 10 mL syringe and filtered using 0.2 μM filters (Minisart Syringe Filters, Sartorius, Germany) and collected onto a 2 mL Eppendorf tube and kept at −20° C. until use. The day of manipulation, the preparation was defrosted at 37° C.
    • Heating at 60° C. for 30 minutes:
    • after incubation at room temperature, the preparations were placed in an autoclave (VWR, USA) and underwent a 20 minutes cycle at 60° C. After cooling down, the treated preparations were centrifuged at 4000 rpm during 10 min (Multifuge 35-R, Heraeus, Germany), 2 mL of the supernatant was collected with a 10 mL syringe and filtered using 0.2 μM filters (Minisart Syringe Filters, Sartorius, Germany) and collected onto a 2 mL Eppendorf tube and kept at −20° C. until use. The day of manipulation, the preparation was defrosted at 37° C.
    • Heating at 119° C. for 20 minutes:
    • after incubation at room temperature, the preparations were placed in an autoclave (VWR, USA) and underwent a 20 minutes sterilization cycle at 119° C. After cooling down, the treated preparations were kept at −20° C. until use. The day of manipulation, the preparation was defrosted at 37° C., centrifuged at 4000 rpm during 10 min (Multifuge 35-R, Heraeus, Germany), 1 mL of the supernatant was collected with a 10 mL syringe and filtered using 0.2 μM filters (Minisart Syringe Filters, Sartorius, Germany) and collected onto a 2 mL Eppendorf tube.

These samples will herein called “processed sample”.

The biological activity of each processed sample was determined by using Staphylococcus aureus strain ATCC 25923, according to the previously described Method C.

The bacteria planktonic growth percentage of 20 processed samples, prepared starting from the herbal compositions after each of the different heating treatment presented before, at 1:10 dilution are given in Table 2-2.

TABLE 2-2 planktonic growth of ATCC 25923 with 20 processed samples at 1:10 dilution prepared starting from the herbal compositions (Mix 1 and Mix 2 of Table 2-1) Sucrose Mix 1 Mix 2 Temperature concentration Processed Processed treatment (g/l) samples samples Cold 100 −4% 48% infusion 75 −3% 52% 50 −4% 31% 25 −4% 31% 0 1% 0% 60° C. 100 0% 82% 30 min 75 4% 83% 50 1% 83% 25 1% 86% 0 −4% 16% 119° C. 100 103% 92% 20 min 75 103% 91% 50 70% 94% 25 103% 99% 0 96% 99%

All processed samples prepared from Mix 1 showed antimicrobial effects on ATCC 25923, only after temperature treatment at 1190, whereas Mix 2 showed limited antimicrobial effects (31 to 52% of inhibition) after cold infusion for all concentration of sucrose except for the condition without sucrose and after all tested temperature treatments (>82% of inhibition) except for the condition with no sucrose addition treated at 60° C., for which the processed sample showed only 16% of planktonic growth inhibition. The temperature treatments enhanced the antimicrobial effects of Mix 2. The optimal temperature treatment among tested treatments for Mix 1 and Mix 2 was 119° C. for 20 minutes.

Sucrose concentration influence on antimicrobial effect depends on the temperature treatment applied. For Mix 1 treated at 119° C. during 20 min, the same antimicrobial effect was obtained with 100, 75, 25 and 0 g/l of sucrose, lower activity was observed with 50 g/l. For Mix 2, the same antimicrobial effect was obtained regardless of sucrose concentration for 119° C. temperature treatment.

The biofilm formation inhibition (IBF) percentage of 20 processed samples, prepared starting from the herbal compositions after each of the different heating treatment presented before, at 1:10 dilution are given in Table 2-3.

TABLE 2-3 biofilm formation inhibition (IBF) of ATCC 25923 with 20 processed samples at 1:10 dilution prepared starting from the herbal compositions (Mix 1 and Mix 2 of Table 2-1) Sucrose Mix1 Mix 2 Temperature concentration Processed Processed treatment (g/l) samples samples Cold infusion 100 34% 74% 75 29% 72% 50 22% 60% 25 30% 55% 0 54% 50%  60° C. 100 48% 91% 30 min 75 34% 94% 50 26% 91% 25 26% 88% 0 48% 78% 119° C. 100 91% 89% 20 min 75 91% 88% 50 75% 89% 25 92% 87% 0 90% 89%

In all tested conditions, processed samples showed some antibiofilm effect.

For Mix 1, limited antibiofilm activity was observed for all concentration of sucrose with cold infusion and with 60° C. temperature treatment (22 to 55% of inhibition). High antibiofilm activity (>90% of inhibition) was observed with 119° C. temperature treatment for all concentrations of sucrose except for 50 g/l which showed 75% of inhibition.

For Mix 2, equivalent antibiofilm activities were obtained regardless of sucrose concentrations for 60° C. and 119° C. temperature treatments (>77% of inhibition). After cold infusion, Mix 2 showed medium to good antibiofilm effect (from 50% to 74% of inhibition) depending on the sucrose concentrations: the more the sucrose concentration was high, the more the preparation was active.

Example 3

In this Example we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus and Staphylococcus epidermidis of 12 mixtures containing 2 to 7 plants.

The dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France).

Herbal compositions comprising from 2 to 7 plant powders were prepared by collecting a fixed volume of plant powder with a calibrated spoon allowing to collect 200 μL of powder

Twelve herbal composition were prepared as below (Table 3-1) according to the previously described Method A.

TABLE 3-1 herbal compositions of processed samples Herbal PA13 PA21 PB12 PA11 PA12 PA22 PA79 compositions (nb spoon) (nb spoon) (nb spoon) (nb spoon) (nb spoon) (nb spoon) (nb spoon) Mix[2P]-1 1 1 Mix[2P]-2 1 1 Mix[2P]-3 1 1 Mix[3P]-1 1 1 1 Mix[4P]-1 1 1 1 1 Mix[4P]-2 1 1 1 1 Mix[4P]-3 1 1 1 1 Mix[5P]-1 1 1 1 1 1 Mix[5P]-2 1 1 1 1 1 Mix[5P]-3 1 1 1 1 1 Mix[6P]-1 1 1 1 1 1 1 Mix[7P]-1 1 1 1 1 1 1 1

Twelve corresponding processed samples were prepared starting from each herbal composition according to the previously described Method B using 20 mL of water containing 100 g/l of sucrose for water extraction and including the centrifugation step before filtration.

The biological activity of the processed sample was determined by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 29213, NCTC 12493, ATCC 33591, ATCC 33592, ATCC 43300, ATCC 700698, ATCC 700699, ATCC 9144 and ATCC BAA-44 and the following Staphylococcus epidermidis strains: ATCC 12228, ATCC 700296, ATCC 49461 and ATCC 14990, according to the previously described Method C.

The minimum inhibitory concentration (MIC) observed with the 12 processed samples is given in Table 3-2 in the form of HID, as well as the average efficacy over all the strains of Staphylococcus aureus and Staphylococcus epidermidis.

TABLE 3-2 HID of the 12 processed samples on 10 strains of Staphylococcus aureus and 4 strains of Staphylococcus epidermidis Mix[2P]-1 Mix[2P]-2 Mix[2P]-3 Mix[3P]-1 Mix[4P]-1 Mix[4P]-2 S. aureus ATCC 25923 10 10 10 10 10 10 ATCC 29213 10 10 0 10 10 10 NCTC 12493 10 10 10 10 10 20 ATCC 33591 10 10 10 10 10 10 ATCC 33592 10 10 10 10 10 10 ATCC 43300 10 10 0 10 10 10 ATCC 700698 10 10 10 10 10 10 ATCC 700699 10 10 10 20 20 20 ATCC 9144 10 10 10 10 10 10 ATCC BAA-44 10 10 10 10 10 20 Average 10.0 10.0 8.0 11.0 11.0 13.0 S. epidermidis ATCC 12228 10 10 10 20 20 20 ATCC 700296 20 20 20 20 20 20 ATCC 49461 10 10 20 20 20 20 ATCC 14990 10 20 10 20 20 20 Average 12.5 15 15 20 20 20 Total 10.7 11.4 10.0 13.6 13.6 15.0 average Mix[4P]-3 Mix[5P]-1 Mix[5P]-2 Mix[5P]-3 Mix[6P]-1 Mix[7P]-1 S. aureus ATCC 25923 10 10 10 20 10 20 ATCC 29213 10 10 10 10 10 10 NCTC 12493 20 20 20 20 20 20 ATCC 33591 10 20 20 20 20 20 ATCC 33592 10 20 20 20 10 20 ATCC 43300 10 10 10 10 10 20 ATCC 700698 10 20 20 20 10 20 ATCC 700699 20 20 20 20 20 20 ATCC 9144 10 20 20 20 10 20 ATCC BAA-44 20 20 20 20 20 20 Average 13.0 17.0 17.0 18.0 14.0 19.0 S. epidermidis ATCC 12228 20 20 20 20 20 40 ATCC 700296 20 20 20 20 20 40 ATCC 49461 20 20 20 20 20 20 ATCC 14990 20 20 20 20 20 20 Average 20 20 20 20 20 30 Total 15.0 17.9 17.9 18.6 15.7 22.1 average

Since in this Example observed HID are in most case between 1:10 and 1:20, 1:20 dilution was chosen as the dilution of reference for the calculation of bacteria planktonic growth inhibition percentage and bioflim formation inhibition percentage. The bacteria planktonic growth inhibition percentage of the 12 processed samples at 1:20 dilution is given in Table 3-3, as well as the average efficacy over all the strains of Staphylococcus aureus and Staphylococcus epidermidis.

TABLE 3-3 planktonic growth inhibition of the 12 processed samples on 10 strains of Staphylococcus aureus and 4 strains of Staphylococcus epidermidis at 1:20 dilution Mix Mix Mix Mix Mix Mix Mix Mix Mix Mix Mix Mix [2P]- [2P]- [2P]- [3P]- [4P]- [4P]- [4P]- [5P]- [5P]- [5P]- [6P]- [7P]- 1 2 3 1 1 2 3 1 2 3 1 1 S. aureus ATCC 25923 12% 12%  9% 11%  20%  11%  20%  34%  41%  35%  21%  96% ATCC 29213 11% 11%  7% 11%  19%  11%  11%  33%  33%  33%  29%  45% NCTC 12493 19%  8%  5% 25%  21% 101%  97%  98% 108% 108%  96% 100% ATCC 33591 12%  8%  8% 12%  19%  16%  34% 104% 107% 111%  83%  98% ATCC 33592 19% 15% 12% 18%  26%  23%  26%  86% 105%  98%  38% 100% ATCC 43300 18% 11% 11% 15%  18%  18%  18%  33%  34%  37%  16%  97% ATCC 700698 11%  8%  8% 11%  11%  11%  16% 107%  97%  73%  40%  98% ATCC 700699 43% 36% 38% 87%  98%  99%  99% 100% 108% 112% 116%  98% ATCC 9144 12%  7%  8% 12%  24%  17%  24%  80%  97%  73%  43%  94% ATCC BAA-44 30% 11% 12% 32%  28%  83% 102% 100% 107% 110% 114% 103% Average 19% 13% 12% 23%  28%  39%  45%  78%  84%  79%  60%  93% S. ATCC 12228 49% 49% 66% 94%  99%  99%  98%  99%  99% 104% 114%  97% epidermidis ATCC 700296 87% 83% 78% 94%  99%  99%  95% 101%  98% 102% 105%  92% ATCC 49461 68% 68% 70% 93% 101% 101%  97% 102% 102% 106% 112%  93% ATCC 14990 38% 71% 61% 92% 103% 100%  95% 102% 102% 106% 107%  93% Average 61% 68% 69% 94% 100% 100%  96% 101% 100% 104% 109%  94% Total average 31% 28% 28% 43%  49%  56%  59%  84%  88%  86%  74%  93%

The biofilm formation inhibition (IBF) percentage of the 12 processed samples at 1:20 dilution is given in Table 3-4, as well as the average efficacy over all the strains of Staphylococcus aureus and Staphylococcus epidermidis. The average efficacy of bacteria planktonic growth inhibition and biofilm formation inhibition percentage over all the tested strains is a good indicator of antimicrobial and antibiofilm efficacy on different bacterial strains.

TABLE 3-4 biofilm formation inhibition (IBF) of the 12 processed samples on 10 strains of Staphylococcus aureus and 4 strains of Staphylococcus epidermidis at 1:20 dilution Mix Mix Mix Mix Mix Mix Mix Mix Mix Mix Mix Mix [2P]- [2P]- [2P]- [3P]- [4P]- [4P]- [4P]- [5P]- [5P]- [5P]- [6P]- [7P]- 1 2 3 1 1 2 3 1 2 3 1 1 S. aureus ATCC 25923 37%  14% 22% 18% 29% 36% 35%  51%  41%  40%  38% 90% ATCC 29213 49%  12% 38% 27% 33% 38% 25%  35%  38%  34%  38% 41% NCTC 12493 49%  −6% 47% 36% 35% 88% 87%  92%  92%  89%  47% 93% ATCC 33591 38% −21% 30% 16% 20% 25% 19%  92%  91%  89%  57% 93% ATCC 33592 51% −10% 26% 25% 43% 47% 31%  60%  78%  70%  43% 91% ATCC 43300 33% −19% 24%  2% 18% 26%  2% −15% −22% −18% −19% 68% ATCC 700698 27%  −8% 20% 10% 25% 23% 13%  62%  61%  39%  24% 88% ATCC 700699 61%  34% 63% 64% 79% 78% 86%  89%  87%  86%  87% 90% ATCC 9144 33% −10% 22% 16% 21% 10% 16%  37%  59%  28%   0% 81% ATCC BAA-44 50%  22% 47% 40% 39% 48% 89%  92%  90%  87%  91% 93% Average 43%   1% 34% 25% 34% 42% 40%  59%  62%  54%  41% 83% S. ATCC 12228 62% −32% 78% 84% 95% 92% 92%  92%  91%  93%  96% 96% epidermidis ATCC 700296 61%  39% 78% 73% 85% 86% 84%  80%  75%  76%  79% 87% ATCC 49461 18%  15% 72% 84% 90% 90% 90%  88%  88%  86%  90% 94% ATCC 14990 68%  39% 68% 88% 94% 91% 92%  90%  90%  89%  91% 90% Average 52%  15% 74% 82% 91% 90% 89%  87%  86%  86%  89% 92% Total average 45%   5% 45% 42% 50% 56% 54%  67%  69%  63%  54% 85%

2-plants mixes extracts showed full antimicrobial activity at 1:10 dilution on the 10 Staphylococcus aureus strains tested except for the Mix[2P]-3 which had no antimicrobial at the tested dilution on 2 of the 10 Staphylococcus aureus strains tested (ATCC 29213 and ATCC 43300) (see Table 3-2).

The three 2-plant mixes extracts showed different antimicrobial activity on the 4 Staphylococcus epidermidis strains tested. Mix[2P]-1 had an HID of 1:10 on 3 strains (ATCC 12228, ATCC 49461 and ATCC 14990) and an HID of 1:20 on ATCC 700296 strain. Mix[2P]-2 had an HID of 1:10 on 2 strains (ATCC 12228 and ATCC 49461) and an HID of 1:20 on the 2 other strains (ATCC 700296 and ATCC 14990). Whereas Mix[2P]-3 had an HID of 1:10 on 2 strains (ATCC 12228 and ATCC 14990) and an HID of 1:20 on the 2 other strains (ATCC 700296 and ATCC 49461) (see Table 3-2).

Mix[3P]-1 obtained from the 3 plants which are included in Mix[2P]-1, Mix[2P]-2 and Mix[2P]-3, showed a wider antimicrobial activity than the 3 two-plant mixes (HID of 1:10 on 9 Staphylococcus aureus strains and HID of 1:20 on ATCC 700699, HID of 1:20 on the 4 Staphylococcus epidermidis tested) (see Table 3-2). The gain of antimicrobial activity is confirmed by the calculation of the planktonic growth inhibition percentage of each plant mixes (see Table 3-3).

3 different plants, PA11, PA12 and PA22, were added to the 3-plant mix Mix[3P]-1 to obtain three 4 plant-mixes extracts, Mix[4P]-1, Mix[4P]-2 and Mix[4P]-3 respectively. These 4-plant mixes extracts showed same HID or better HID than the 3-plant mix extract Mix[3P]-1 (see Table 3-2), and higher planktonic growth inhibition percentage: an average of 28% to 45% is obtained with 4-plant mixes extracts on the 10 Staphylococcus aureus strains compare to 23% with the 3-plant mix extract (see Table 3-3). They also showed higher biofilm formation inhibition percentage: an average of 34% to 42% is obtained with 4-plant mixes extracts on the 10 Staphylococcus aureus strains compare to 25% with the 3-plant mix extract (see Table 3-4).

5-plant mixes were prepared by adding 2 plants among the 3 selected additional plants. All the three 5-plant mixes extracts showed higher antimicrobial and antibiofilm activity than the 4-plant mixes extracts (see Tables 3-2, 3-3 and 3-4).

Mix[6P]-1 consists in the 6 plants which were used for the preparation of the previously tested processed samples. Interestingly, the 6-plant mix extract showed less antimicrobial and antibiofilm activity than 5-plant mixes extracts but higher antimicrobial effect and higher to equivalent antibiofilm effects than 4-plant mixes extracts. Antimicrobial and antibiofilm activity was recovered and enhanced by the addition of a new plant (PA79) (see Tables 3-2, 3-3 and 3-4).

Increasing the number of plants in the mixes allowed to prepare corresponding water extracts having enhanced antimicrobial and antibiofilm activity and increased the number of strains, in which one or both these activities can be identified.

Example 4

In this Example we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus of 196 water extract obtained from herbal compositions including at least 2 plants among the 3 plants described in Example 1 and additional plants to form mixtures of 3 to 10 plants.

The dried plants of pharmaceutical grade were obtained from “Pharmacie Fontgiève” (Clermont-Ferrand, France, plants PA00 to PB12) and “Pharmacie St Herem” (Clermont-Ferrand, France, plants PB13 to PD08).

The 4 reference herbal compositions Mix[2P]-1, Mix[2P]-2, Mix[2P]-3, Mix[3P]-1 and the 196 new herbal compositions were prepared according to the previously described Method A and had the content reported in List 4-1.

List 4-1: Herbal Compositions of Processed Samples

Mix[2P]-1: PA13; PA21;

Mix[2P]-2: PA21; PB12;

Mix[2P]-3: PA13; PB12;

Mix[3P]-1: PA13; PA21; PB12;

Mix[3P]-2: PA11; PA13; PA21;

Mix[3P]-3: PA21; PB12; PC63;

Mix[3P]-4: PA13; PB12; PC63;

Mix[3P]-5: PA13; PA21; PC63;

Mix[3P]-6: PA13; PA21; PB12;

Mix[3P]-7: PA21; PB12; PC29;

Mix[3P]-8: PA13; PB12; PC29;

Mix[3P]-9: PA13; PA21; PB12;

Mix[4P]-1: PA11; PA13; PA21; PB12;

Mix[4P]-2: PA12; PA13; PA21; PB12;

Mix[4P]-3: PA13; PA21; PA22; PB12;

Mix[4P]-4: PA11; PA13; PA21; PA22;

Mix[4P]-5: PA13; PA21; PB12; PC63;

Mix[4P]-6: PA11; PA12; PA21; PB12;

Mix[4P]-7: PA11; PA12; PA13; PB12;

Mix[4P]-8: PA11; PA13; PA21; PB12;

Mix[4P]-9: PA12; PA13; PA21; PB12;

Mix[4P]-10: PA13; PA21; PB12; PC29;

Mix[5P]-1: PA12; PA13; PA21; PA22; PB12;

Mix[5P]-2: PA11; PA13; PA21; PA22; PB12;

Mix[5P]-3: PA11; PA12; PA13; PA21; PB12;

Mix[5P]-4: PA11; PA12; PA21; PA22; PB12;

Mix[5P]-5: PA11; PA12; PA13; PA22; PB12;

Mix[5P]-6: PA11; PA12; PA13; PA21; PA22;

Mix[5P]-7: PA13; PA21; PA22; PB12; PC63;

Mix[5P]-8: PA13; PA21; PB12; PC29; PC63;

Mix[6P]-1: PA11; PA12; PA13; PA21; PA22; PB12;

Mix[6P]-2: PA12; PA13; PA21; PA22; PB12; PC63;

Mix[6P]-3: PA11; PA13; PA21; PA22; PB12; PC63;

Mix[6P]-4: PA11; PA12; PA13; PA21; PB12; PC63;

Mix[6P]-5: PA12; PA13; PA21; PA22; PB12; PC63;

Mix[6P]-6: PA11; PA13; PA21; PA22; PB12; PC63;

Mix[6P]-7: PA11; PA12; PA21; PA22; PB12; PC63;

Mix[6P]-8: PA11; PA12; PA13; PA22; PB12; PC63;

Mix[6P]-9: PA11; PA12; PA13; PA21; PB12; PC63;

Mix[6P]-10: PA11; PA12; PA13; PA21; PA22; PC63;

Mix[6P]-11: PA11; PA12; PA13; PA21; PA22; PB12;

Mix[7P]-1: PA11; PA12; PA13; PA21; PA22; PB12; PA79;

M[7P]-2: PA11; PA12; PA13; PA21; PA22; PB12; PB49;

M[7P]-3: PA11; PA12; PA13; PA21; PA22; PB12; PC29;

M[7P]-4: PA11; PA12; PA13; PA21; PA22; PB12; PC63;

M[9P]-11: PA12; PA13; PA21; PA22; PA79; PB12; PB49; PC29; PC63;

M[9P]-12: PA11; PA13; PA21; PA22; PA79; PB12; PB49; PC29; PC63;

M[9P]-13: PA11; PA12; PA21; PA22; PA79; PB12; PB49; PC29; PC63;

M[9P]-14: PA11; PA12; PA13; PA22; PA79; PB12; PB49; PC29; PC63;

M[9P]-15: PA11; PA12; PA13; PA21; PA79; PB12; PB49; PC29; PC63;

M[9P]-16: PA11; PA12; PA13; PA21; PA22; PB12; PB49; PC29; PC63;

M[9P]-17: PA11; PA12; PA13; PA21; PA22; PA79; PB49; PC29; PC63;

M[9P]-18: PA11; PA12; PA13; PA21; PA22; PA79; PB12; PC29; PC63;

M[9P]-19: PA11; PA12; PA13; PA21; PA22; PA79; PB12; PB49; PC63;

M[9P]-20: PA11; PA12; PA13; PA21; PA22; PA79; PB12; PB49; PC29;

M[10P]-40: PA11; PA13; PA14; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

M[10P]-44: PA13; PA40; PA41; PA62; PA75; PB18; PC22; PC63; PC64; PD05;

M[10P]-71: PA21; PA85; PB14; PB18; PB85; PC19; PC61; PC63; PD05; PD08;

M[10P]-75: PA02; PA10; PA12; PA13; PA18; PA21; PA27; PA79; PB86; PC26;

M[10P]-84: PA10; PA21; PA27; PB12; PB15; PC26; PC68; PD05; PD06; PD08;

M[10P]-98: PA11; PA12; PA13; PA14; PA20; PA21; PA27; PB16; PB18; PD06;

M[10P]-102: PA12; PA13; PA19; PA21; PA41; PA79; PB10; PC26; PC61; PC64;

M[10P]-103: PA13; PA17; PA21; PA24; PB08; PB49; PB85; PC15; PC29; PD05;

M[10P]-105: PA10; PA11; PA12; PA18; PA19; PA21; PA22; PB12; PC63; PD08;

M[10P]-109: PA10; PA12; PA13; PA21; PA27; PA75; PB14; PB16; PC45; PC63;

M[10P]-112: PA08; PA13; PA15; PA17; PA18; PA21; PA27; PB18; PB41; PB81;

M[10P]-115: PA13; PA20; PA21; PA22; PA62; PA79; PB08; PB19; PC15; PC61;

M[10P]-119: PA11; PA13; PA14; PA24; PB12; PB41; PB49; PC51; PD06; PD08;

M[10P]-123: PA10; PA13; PA15; PA20; PA21; PA75; PB12; PB18; PB49; PC63;

M[10P]-124: PA08; PA13; PA21; PA24; PA27; PB08; PB41; PB49; PC64; PD05;

M[10P]-125: PA13; PA14; PA19; PA21; PA75; PB12; PC15; PC26; PC61; PC63;

M[10P]-129: PA08; PA12; PA18; PA21; PA41; PB08; PB85; PC63; PC64; PD08;

M[10P]-130: PA12; PA13; PA21; PA24; PA27; PB10; PB41; PB49; PC15; PC61;

M[10P]-132: PA12; PA20; PA21; PA24; PC15; PC26; PC61; PC63; PC64; PD08;

M[10P]-134: PA21; PA22; PA24; PA27; PB10; PB12; PB49; PC15; PC29; PD05;

M[10P]-136: PA10; PA11; PA12; PA13; PA18; PA21; PB16; PB18; PC26; PC29;

M[10P]-140: PA13; PA15; PA21; PA24; PA62; PA79; PB10; PB12; PC63; PC64;

M[10P]-142: PA11; PA12; PA13; PA19; PA21; PB12; PB85; PC15; PC26; PC64;

M[10P]-144: PA13; PA15; PA21; PA24; PA75; PB49; PC51; PC61; PC64; PD05;

M[10P]-145: PA12; PA13; PA17; PA20; PA21; PA79; PB08; PB16; PB85; PC63;

M[10P]-148: PA10; PA13; PA20; PA24; PB08; PB12; PB49; PC26; PC51; PC64;

M[10P]-149: PA13; PA14; PA15; PA19; PA21; PB08; PB10; PB41; PC15; PC61;

M[10P]-150: PA10; PA12; PA13; PA14; PA21; PA62; PB10; PC26; PC64; PD06;

M[10P]-151: PA10; PA13; PA21; PA22; PA24; PA41; PB49; PC15; PC61; PC63;

M[10P]-152: PA11; PA12; PA13; PA15; PA21; PA27; PA75; PB12; PB49; PB85;

M[10P]-154: PA11; PA12; PA13; PA21; PA22; PA79; PB12; PB49; PC29; PC63;

M[10P]-156: PA15; PA21; PA22; PB10; PB85; PC26; PC29; PC61; PC63; PC64;

M[10P]-157: PA11; PA13; PA15; PA19; PA21; PB18; PB41; PC15; PC51; PD08;

M[10P]-158: PA10; PA17; PA21; PA24; PA27; PA41; PB49; PC26; PC61; PC63;

M[10P]-160: PA10; PA12; PA13; PA15; PA18; PA20; PA21; PA24; PB12; PC26;

M[10P]-162: PA12; PA13; PA21; PA27; PA79; PB12; PB16; PB18; PC63; PC64;

M[10P]-164: PA12; PA13; PA15; PA19; PA21; PA27; PB08; PB12; PB49; PD05;

M[10P]-165: PA13; PA15; PA22; PA75; PA79; PB12; PB18; PC15; PC26; PC29;

M[10P]-167: PA12; PA13; PA75; PB08; PB12; PB85; PC15; PC51; PC61; PC63;

M[10P]-168: PA11; PA13; PA17; PA21; PA27; PA62; PA79; PB16; PB49; PC64;

M[10P]-169: PA12; PA17; PA21; PA24; PA75; PA79; PB10; PB12; PB85; PC15;

M[10P]-170: PA11; PA13; PA15; PA20; PA21; PA22; PC26; PC29; PC63; PC64;

M[10P]-172: PA13; PA20; PA21; PA75; PB10; PB12; PB18; PB49; PC29; PC61;

M[10P]-173: PA10; PA12; PA13; PA14; PA19; PA20; PA21; PA79; PC15; PC63;

M[10P]-174: PA11; PA12; PA13; PA27; PA62; PB12; PB16; PB49; PB85; PC26;

M[10P]-175: PA11; PA12; PA13; PA21; PA22; PA75; PB10; PB49; PC15; PC63;

M[10P]-176: PA11; PA13; PA21; PA24; PA62; PB12; PC15; PC26; PC61; PC64;

M[10P]-178: PA11; PA14; PA15; PA18; PA19; PA21; PA22; PB16; PC15; PC63;

M[10P]-180: PA08; PA11; PA12; PA13; PA15; PA17; PA21; PA62; PB12; PC63;

M[10P]-181: PA13; PA19; PA21; PA22; PA79; PB08; PB10; PB18; PC15; PC29;

M[10P]-182: PA11; PA15; PA20; PA21; PA22; PA75; PB12; PC29; PC63; PC64;

M[10P]-183: PA11; PA12; PA13; PA27; PA79; PB16; PB18; PB49; PC15; PC63;

M[10P]-184: PA11; PA13; PA15; PA20; PA21; PA22; PA27; PA75; PB12; PC29;

M[10P]-186: PA11; PA15; PA19; PA20; PA21; PA22; PA27; PB10; PB12; PB85;

M[10P]-188: PA11; PA12; PA13; PA19; PA21; PA75; PA79; PB49; PC15; PC63;

M[10P]-190: PA11; PA13; PA21; PA22; PA27; PA75; PB10; PB12; PB49; PC63;

M[10P]-191: PA13; PA19; PA20; PA21; PA79; PB10; PB18; PB85; PC29; PC61;

M[10P]-192: PA12; PA13; PA19; PA21; PA27; PA75; PB12; PB49; PC63; PC64;

M[10P]-193: PA13; PA15; PA20; PA21; PA79; PB16; PC15; PC26; PC29; PC61;

M[10P]-194: PA11; PA17; PA21; PA22; PA27; PB12; PB49; PC15; PC63; PC64;

M[10P]-196: PA11; PA12; PA13; PA15; PA21; PA75; PB10; PB18; PC51; PC63;

M[10P]-197: PA11; PA12; PA13; PA15; PA21; PA24; PA79; PB49; PC61; PC64;

M[10P]-198: PA12; PA15; PA21; PA22; PB12; PB16; PB85; PC15; PC26; PC29;

M[10P]-199: PA11; PA12; PA13; PA15; PA21; PA75; PB12; PC15; PC63; PC64;

M[10P]-201: PA11; PA13; PA20; PA21; PA22; PA27; PA75; PB12; PB49; PC64;

M[10P]-202: PA11; PA21; PA75; PB10; PB12; PB61; PC26; PC29; PC42; PC63;

M[10P]-205: PA11; PA13; PA19; PA21; PA22; PA27; PA75; PB12; PB41; PC63;

M[10P]-207: PA11; PA13; PA21; PA75; PB10; PB12; PB41; PB51; PC45; PC63;

M[10P]-209: PA12; PA13; PA21; PA24; PA75; PB12; PB16; PB49; PB60; PC15;

M[10P]-210: PA13; PA15; PA20; PA22; PA27; PB08; PB10; PB12; PB18; PC63;

M[10P]-212: PA11; PA13; PA21; PA22; PA27; PA75; PB12; PB16; PC15; PC63;

M[10P]-214: PA11; PA12; PA15; PA21; PA22; PA75; PB12; PB49; PC15; PC61;

M[10P]-215: PA11; PA12; PA13; PA21; PA25; PB12; PC02; PC29; PC51; PC64;

M[10P]-216: PA11; PA12; PA13; PA21; PA27; PA75; PA85; PB18; PB49; PC49;

M[10P]-217: PA18; PA20; PA21; PA22; PA75; PB10; PB12; PB16; PB85; PC64;

M[10P]-218: PA10; PA13; PA15; PA20; PA22; PA27; PB18; PB49; PC51; PC63;

M[10P]-219: PA12; PA13; PA15; PA19; PA21; PA22; PA75; PB10; PB12; PC26;

M[10P]-220: PA11; PA12; PA13; PA19; PA21; PA22; PA27; PB12; PB49; PC63;

M[10P]-221: PA13; PA15; PA19; PA20; PA21; PA41; PA75; PB12; PC15; PC60;

M[10P]-222: PA05; PA13; PA21; PA22; PA75; PA79; PB49; PC26; PC29; PC63;

M[10P]-223: PA11; PA12; PA13; PA22; PA25; PA75; PB12; PB49; PC61; PC64;

M[10P]-225: PA12; PA13; PA21; PA27; PB10; PB12; PC29; PC63; PC64; PD08;

M[10P]-226: PA11; PA12; PA21; PA39; PA79; PB18; PB49; PB85; PC61; PC63;

M[10P]-227: PA11; PA12; PA15; PA20; PA21; PA22; PB12; PB42; PC15; PC26;

M[10P]-229: PA13; PA20; PA62; PA79; PB12; PB46; PB85; PC29; PC64; PD08;

M[10P]-230: PA11; PA12; PA13; PA21; PA22; PA75; PB10; PB12; PB58; PC61;

M[10P]-232: PA12; PA13; PA15; PA22; PA27; PA83; PB12; PB49; PC26; PC63;

M[10P]-235: PA12; PA13; PA15; PA20; PA21; PA22; PC29; PC51; PC63; PC65;

M[10P]-236: PA12; PA13; PA20; PA21; PA27; PA75; PB50; PB85; PC29; PC64;

M[10P]-237: PA11; PA13; PA15; PA21; PA27; PA75; PB10; PB12; PC63; PC70;

M[10P]-239: PA10; PA11; PA12; PA20; PA21; PB10; PB41; PB49; PC63; PD08;

M[10P]-240: PA11; PA12; PA15; PA21; PA27; PA79; PB46; PC29; PC51; PC63;

M[10P]-241: PA11; PA14; PA20; PA21; PA22; PA75; PB12; PB18; PC15; PC29;

M[10P]-242: PA11; PA12; PA13; PA21; PA22; PA79; PB18; PB49; PC51; PC63;

M[10P]-244: PA11; PA12; PA13; PA20; PA22; PA27; PB10; PB12; PC63; PC64;

M[10P]-246: PA11; PA12; PA13; PA21; PA61; PA79; PB10; PB12; PB18; PB85;

M[10P]-247: PA11; PA12; PA21; PA27; PB16; PB49; PC21; PC51; PC61; PC63;

M[10P]-248: PA11; PA13; PA14; PA19; PA21; PA75; PA79; PB14; PB49; PC15;

M[10P]-250: PA11; PA12; PA15; PA20; PA21; PA41; PA75; PB49; PC26; PC63;

M[10P]-251: PA12; PA13; PA21; PA22; PA27; PA79; PB10; PB12; PB13; PB16;

M[10P]-252: PA11; PA13; PA20; PA21; PA44; PB08; PB12; PB18; PB88; PD06;

M[10P]-253: PA10; PA11; PA12; PA13; PA15; PA21; PA22; PA27; PA79; PC63;

M[10P]-255: PA11; PA13; PA21; PA75; PB12; PB18; PB49; PC15; PC26; PC63;

M[10P]-256: PA11; PA12; PA13; PA21; PA22; PA23; PA75; PA79; PC29; PC64;

M[10P]-257: PA13; PA20; PA21; PA27; PB12; PB49; PB83; PC29; PC63; PD00;

M[10P]-258: PA12; PA13; PA20; PA21; PA22; PA75; PB10; PB12; PC15; PC63;

M[10P]-262: PA11; PA12; PA21; PA31; PA75; PB49; PB85; PC61; PC63; PC64;

M[10P]-264: PA21; PA24; PA27; PA31; PA79; PB10; PB18; PB49; PC15; PC63;

M[10P]-267: PA11; PA12; PA21; PA22; PA75; PC29; PC51; PC52; PC61; PC63;

M[10P]-268: PA11; PA12; PA13; PA15; PA27; PA75; PB10; PB12; PB45; PC64;

M[10P]-269: PA13; PA20; PA21; PA22; PA79; PB10; PB11; PB49; PC61; PC63;

M[10P]-270: PA11; PA12; PA13; PA15; PA21; PA75; PB12; PB14; PB18; PC49;

M[10P]-271: PA11; PA13; PA20; PA22; PA27; PA45; PB08; PB12; PB49; PC64;

M[10P]-272: PA13; PA21; PA24; PA79; PB10; PB12; PB41; PC15; PC29; PC63;

M[10P]-273: PA11; PA15; PA21; PA22; PA27; PA75; PB12; PB14; PC64; PD00;

M[10P]-274: PA12; PA13; PA21; PA22; PB12; PB49; PC26; PC51; PC63; PC64;

M[10P]-278: PA13; PA15; PA21; PA22; PA75; PA79; PB10; PB12; PB49; PC63;

M[10P]-280: PA11; PA12; PA15; PA21; PB08; PB12; PB18; PB49; PB58; PC51;

M[10P]-281: PA11; PA12; PA20; PA21; PA22; PA27; PA29; PA75; PB16; PC63;

M[10P]-282: PA10; PA13; PA21; PA22; PA27; PA79; PB12; PC15; PC29; PC61;

M[10P]-284: PA11; PA13; PA20; PA21; PA22; PA27; PA95; PC15; PC29; PC63;

M[10P]-285: PA11; PA13; PA14; PA21; PA79; PB10; PB12; PB18; PB49; PB82;

M[10P]-286: PA11; PA12; PA13; PA14; PA20; PA21; PA79; PC51; PC64; PC82;

M[10P]-287: PA12; PA13; PA19; PA20; PA75; PB10; PB12; PC26; PC63; PD08;

M[10P]-289: PA12; PA13; PA15; PA19; PA20; PA21; PB10; PB12; PC29; PC51;

M[10P]-290: PA11; PA12; PA13; PA22; PA27; PA75; PB85; PC29; PC63; PDO7;

M[10P]-292: PA12; PA13; PA15; PA75; PA79; PB10; PB12; PC29; PC45; PC63;

M[10P]-293: PA00; PA11; PA12; PA13; PA21; PA27; PA75; PB18; PB85; PC26;

M[10P]-294: PA12; PA13; PA15; PA21; PA22; PA79; PB12; PB49; PC51; PC63;

M[10P]-296: PA11; PA13; PA15; PA17; PA21; PA62; PA79; PB10; PB12; PC63;

M[10P]-300: PA12; PA13; PA17; PA48; PB10; PB12; PB16; PC26; PC29; PC63;

M[10P]-301: PA11; PA12; PA13; PA20; PA21; PA75; PB08; PC15; PC35; PC51;

M[10P]-302: PA11; PA12; PA13; PA21; PA70; PB12; PB16; PB18; PC26; PC63;

M[10P]-303: PA11; PA12; PA15; PA21; PA75; PA79; PB10; PB12; PC29; PC64;

M[10P]-307: PA12; PA13; PA21; PB10; PB12; PB18; PB78; PC51; PC63; PD08;

M[10P]-308: PA17; PA21; PA22; PA27; PB12; PB49; PB85; PC15; PC26; PC29;

M[10P]-309: PA13; PA22; PA27; PA53; PA62; PA75; PB10; PC51; PC63; PDO6;

M[10P]-311: PA12; PA15; PA19; PA21; PA79; PB12; PB49; PC26; PC63; PC83;

M[10P]-312: PA00; PA11; PA13; PA21; PA22; PA29; PA75; PB10; PB85; PC29;

M[10P]-313: PA13; PA15; PA21; PA22; PA27; PA75; PB49; PC51; PC61; PC63;

M[10P]-314: PA08; PA12; PA13; PA21; PB10; PB12; PB49; PC15; PC32; PC64;

M[10P]-315: PA11; PA13; PA21; PA47; PA75; PB16; PB83; PC15; PC51; PC63;

M[10P]-317: PA11; PA12; PA19; PA21; PA22; PA79; PB10; PB16; PC29; PC63;

M[10P]-319: PA12; PA13; PA22; PA75; PA79; PA98; PB08; PB10; PB85; PC63;

M[10P]-321: PA11; PA12; PA13; PA20; PA21; PA22; PB49; PB85; PC55; PC63.

The corresponding water extracts (or processed samples) of such herbal compositions were prepared according to the previously described Method B using 20 mL of water containing 100 g/l of sucrose for water extraction and including centrifugation step before filtration.

The biological activity of the processed sample was determined by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213 and ATCC 14775, with the following final dilutions of processed samples: 1:10, 1:20, 1:40 and 1:80 for processed samples containing less than 10 spoons of plant powder (M[2P] to M[9P]) or 1:20, 1:63 and 1:200 for processed samples containing 10 spoons of plant powder (M[10P]), according to the previously described Method C.

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from the 2 plants of Mix[2P]-1 at 1:20 dilution is given in Table 4-1.

TABLE 4-1 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[2P]-1 at 1:20 dilution Planktonic Biofilm growth formation inhibition inhibition (A) Mix[2P]-1 22% 36% M[10P]-124 28% 47% M[10P]-236 24% 50% M[10P]-193 27% 54% M[10P]-286 45% 38% Mix[5P]-6 39% 45% M[10P]-197 32% 55% M[10P]-112 40% 49% M[10P]-157 41% 52% M[10P]-136 39% 55% M[10P]-144 45% 52% M[10P]-191 39% 57% M[10P]-168 44% 53% M[10P]-115 44% 54% M[10P]-98 48% 51% M[10P]-102 51% 54% M[10P]-150 49% 59% M[10P]-103 51% 58% M[10P]-130 57% 61% M[10P]-149 56% 63% M[10P]-181 65% 70% (B) Mix[2P]-1 22% 36% M[10P]-75 23% 40% M[10P]-216 22% 41% M[10P]-256 19% 47% M[10P]-248 14% 48% (C) Mix[2P]-1 22% 36% Mix[3P]-2 27% 26% Mix[4P]-4 27% 29% M[10P]-293 28% 32% M[10P]-301 28% 28% M[10P]-312 40% 37%

20 processed samples obtained from herbal compositions containing 5 or 10 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[2P]-1 (Table 4-1 A). Four 10 plant mixes extracts were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 4-1 B). Five processed samples containing 3, 4 or 10 plants were found to have higher antimicrobial activity and equivalent or lower antibiofilm activity (Table 4-1 C).

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions containing the 2 plants of Mix[2P]-2 at 1:20 dilution is given in Table 4-2.

TABLE 4-2 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[2P]-2 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antibiofilm activity and equivalent or lower antimicrobial activity. (C) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity. Planktonic Biofilm growth formation inhibition inhibition (A) Mix[2P]-2  12% −3% M[10P]-308  16% 13% Mix[3P]-7  23%  9% M[10P]-264  15% 29% M[10P]-317  36%  9% M[10P]-217  17% 28% M[10P]-280  21% 27% Mix[6P]-7  36% 16% M[10P]-267  17% 36% Mix[3P]-3  27% 28% M[10P]-247  16% 40% M[10P]-214  23% 33% M[10P]-303  35% 22% M[10P]-281  17% 42% M[10P]-273  19% 41% Mix[5P]-4  34% 27% M[10P]-198  16% 46% M[10P]-241  26% 38% M[10P]-132  23% 41% M[10P]-239  28% 44% M[10P]-262  25% 48% M[10P]-129  28% 46% M[10P]-186  31% 44% M[10P]-311  40% 38% M[10P]-134  32% 48% M[10P]-182  40% 44% M[10P]-178  39% 45% M[10P]-202  37% 50% M[10P]-156  40% 48% M[10P]-194  41% 49% M[10P]-105  47% 44% M[10P]-240  42% 61% M[10P]-169  53% 57% M[9P]-13 100% 86% (B) Mix[2P]-2  12% −3% M[10P]-84   9% 27% M[10P]-158  12% 30% M[10P]-071  11% 35% M[10P]-227  12% 42% M[10P]-250  11% 44% M[10P]-226  13% 51% (C) Mix[2P]-2  12% −3% Mix[4P]-6  19% −5%

33 processed samples obtained from herbal compositions containing 3, 5, 9 or 10 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[2P]-2 (Table 4-2 A). Six 10 plant mixes extracts were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 4-2 B). One 4 plant mix extract was found to have higher antimicrobial activity and equivalent antibiofilm activity (Table 4-2 C).

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions containing the 2 plants of Mix[2P]-3 at 1:20 dilution is given in Table 4-3.

TABLE 4-3 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[2P]-3 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity. Planktonic Biofilm growth formation inhibition inhibition (A) Mix[2P]-3 12% 28% M[10P]-44 15% 35% M[10P]-271 13% 47% M[10P]-218 19% 42% M[10P]-268 16% 46% Mix[3P]-4 17% 47% M[10P]-210 22% 42% M[10P]-165 22% 42% Mix[5P]-5 29% 36% M[10P]-287 34% 31% M[10P]-148 28% 41% M[10P]-290 31% 39% M[10P]-229 15% 56% M[6P]-8 34% 40% M[10P]-319 38% 37% M[10P]-244 21% 56% M[10P]-223 23% 55% M[10P]-232 25% 61% M[10P]-119 42% 48% M[10P]-183 39% 51% M[10P]-292 47% 48% M[10P]-174 47% 59% M[10P]-167 80% 71% M[9P]-14 90% 83% (B) Mix[2P]-3 12% 28% Mix[4P]-7 17%  5% Mix[3P]-8 19% 14% M[10P]-300 20% 15% M[10P]-309 33%  2%

23 processed samples obtained from herbal compositions containing 3, 5, 9 or 10 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[2P]-3 (Table 4-3 A). Four processed samples obtained from herbal compositions containing 3, 4 or 10 plants were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 4-3 B).

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions containing the 3 plants of Mix[3P]-1 at 1:20 dilution is given in Table 4-4.

TABLE 4-4 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[3P]-1 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antibiofilm activity and equivalent or lower antimicrobial activity. (C) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity. Planktonic Biofilm growth formation inhibition inhibition (A) Mix[3P]-1  31% 15% Mix[4P]-8  37% 20% Mix[6P]-10  33% 25% M[10P]-314  34% 24% Mix[4P]-2  37% 22% Mix[6P]-11  35% 27% Mix[4P]-3  41% 24% M[10P]-315  39% 27% M[10P]-284  41% 31% M[10P]-321  37% 39% M[10P]-109  36% 43% M[10P]-302  44% 35% Mix[5P]-8  43% 40% M[6P]-9  51% 35% M[10P]-160  35% 52% M[10P]-212  43% 45% M[10P]-274  38% 51% M[10P]-246  34% 56% M[10P]-205  42% 48% M[10P]-285  52% 39% M[10P]-201  37% 54% M[10P]-222  32% 59% Mix[4P]-10  52% 40% M[10P]-230  38% 54% M[10P]-220  43% 50% Mix[5P]-2  51% 42% Mix[5P]-1  50% 44% M[10P]-215  38% 55% Mix[6P]-6  56% 38% M[10P]-235  40% 54% M[10P]-207  43% 53% Mix[5P]-3  51% 48% M[10P]-307  58% 43% M[10P]-289  60% 41% M[10P]-272  40% 62% M[10P]-184  48% 55% M[10P]-151  48% 57% M[10P]-123  51% 55% M[6P]-1  60% 50% M[6P]-5  61% 51% M[7P]-3  61% 54% M[10P]-192  57% 60% M[10P]-164  58% 59% M[7P]-1  62% 57% M[10P]-296  63% 60% M[6P]-4  73% 51% Mix[5P]-7  64% 60% M[6P]-2  68% 57% M[10P]-225  60% 66% M[10P]-188  65% 61% Mix[4P]-5  61% 66% M[10P]-125  66% 62% M[10P]-173  65% 67% M[10P]-237  69% 63% M[7P]-3  71% 64% M[10P]-294  71% 65% M[10P]-199  70% 69% M[10P]-142  71% 69% Mix[6P]-3  72% 68% M[10P]-145  72% 69% M[10P]-180  72% 70% M[10P]-140  72% 71% M[10P]-162  73% 70% M[10P]-176  74% 70% M[10P]-172  72% 73% M[10P]-152  71% 74% M[10P]-40  77% 68% M[7P]-4  72% 76% M[10P]-170  80% 74% M[10P]-196  78% 77% M[10P]-190  82% 76% M[10P]-154  80% 79% M[10P]-175  84% 77% M[9P]-20  93% 81% M[9P]-17  95% 84% M[9P]-19  95% 86% M[9P]-11  96% 91% M[9P]-18  98% 89% M[9P]-12 103% 91% M[9P]-15 106% 88% M[9P]-16 106% 91% (B) Mix[3P]-1  31% 15% Mix[3P]-6  16% 20% Mix[4P]-9  30% 21% Mix[3P]-9  19% 23% M[10P]-282  24% 26% Mix[3P]-5  29% 36% M[10P]-269  14% 37% M[10P]-278  18% 40% M[10P]-219  30% 42% M[10P]-255  20% 43% M[10P]-270  15% 43% M[10P]-251  13% 44% M[10P]-258  20% 44% M[10P]-209  21% 45% M[10P]-221  17% 49% M[10P]-253  21% 49% M[10P]-252  18% 50% M[10P]-257  18% 51% M[10P]-242  29% 54% (C) Mix[3P]-1  31% 15% Mix[4P]-1  37% 15% M[10P]-313  38% 12%

80 processed samples obtained from herbal compositions containing 4, 5, 6, 7, 9 or 10 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[3P]-1 (Table 4-4 A). 18 processed samples obtained from herbal compositions containing 3, 4, or 10 plants were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 4-4 B). Two processed samples obtained from herbal compositions 4 and 10 plants were found to have higher antimicrobial activity and equivalent antibiofilm activity (Table 4-4 C).

Comparative Example 2

As a comparative Example, we illustrate several 3 or 10 plants mixes extracts obtained from herbal compositions containing only one plant among the 3 plants of Example 1 (PA13, PA21, PB12), which have been found to have none or poor antimicrobial and antibiofilm activities against Staphylococcus aureus.

Ten herbal compositions, whose content is reported in List 4-8, were prepared according to the previously described Method A.

List 4-8: Herbal Compositions of Processed Samples

M[3P]-31: PA21; PA05; PB06;

M[3P]-35: PA21; PA76; PB50;

M[3P]-37: PA21; PB06; PB50;

M[3P]-43: PB12; PA05; PC34;

M[3P]-45: PB12; PA76; PB50;

M[10P]-65: PA01; PA08; PA25; PA29; PA37; PB12; PB50; PC15; PC37; PD05;

M[10P]-70: PA10; PA16; PA21; PA25; PA37; PA75; PB18; PB45; PC04; PC57;

M[10P]-72: PA08; PA21; PA62; PA70; PA85; PB19; PB31; PB45; PB49; PC26;

M[10P]-89: PA10; PA13; PA18; PA19; PA37; PA41; PA75; PB49; PC21; PC67;

M[10P]-306: PA15; PA21; PA22; PA62; PA75; PB18; PB49; PB85; PC49; PC61;

Ten water extracts (or processed samples) were prepared starting from the corresponding herbal compositions.

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains for such processed samples at 1:20 dilution were determined according to the previously reported Method C and are given in Table 4-5.

TABLE 4-5 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed samples at 1:20 dilution. Planktonic Biofilm growth formation inhibition inhibition Mix[2P]-1 22% 36% Mix[2P]-2 12% −3% Mix[2P]-3 12% 28% M[3P]-45 −4% −5% M[3P]-35  0% −8% M[3P]-37  2% −9% M[3P]-31  2% −9% M[3P]-43  1% −1% M[10P]-72  3%  1% M[10P]-70  1% 15% M[10P]-306 10%  6% M[10P]-65  3% 17% M[10P]-89 10% 27%

All tested processed samples, obtained from herbal compositions composed of 3 or 10 plants, which included one plant among the 3 plants of Example 1 (PA13, PA21, PB12), showed lower antimicrobial activity than the three 2 plant mixes extracts of reference Mix[2P]-1 (obtained from PA13 and PA21), Mix[2P]-2 (obtained from PA21 and PB12) and Mix[2P]-3 (obtained from PA13 and PB12) and lower antibiofilm activity than Mix[2P]-1 and Mix[2P]-3.

Example 5

In this Example, we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus of 390 mixtures extracts obtained from herbal compositions including at least 2 plants among the 3 plants described in Example 1 (cf Table 1-1) and additional plants to assemble mixtures of 9 to 20 plants.

The dried plants of pharmaceutical grade were obtained from “Pharmacie Fontgiève” (Clermont-Ferrand, France, plants PA00 to PB12) and “Pharmacie St Herem” (Clermont-Ferrand, France, plants PB13 to PD02).

Herbal compositions containing from 9 to 20 plant powders were prepared according to the previously described Method A.

The 4 reference herbal compositions Mix[2P]-1, Mix[2P]-2, Mix[2P]-3, Mix[3P]-1 and 390 new herbal compositions were prepared having the content showed below (List 5-3).

List 5-3: Herbal Compositions of Processed Samples

Mix[2P]-1: PA13; PA21;

Mix[2P]-2: PA21; PB12;

Mix[2P]-3: PA13; PB12;

Mix[3P]-1: PA13; PA21; PB12;

M[09P]-1: PA13; PA14; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

M[09P]-2: PA11; PA14; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

M[09P]-3: PA11; PA13; PA20; PA21; PA23; PA67; PB12; PC27; PC60;

M[09P]-4: PA11; PA13; PA14; PA21; PA23; PA67; PB12; PC27; PC60;

M[09P]-6: PA11; PA13; PA14; PA20; PA23; PA67; PB12; PC27; PC60;

M[09P]-6: PA11; PA13; PA14; PA20; PA21; PA67; PB12; PC27; PC60;

M[09P]-7: PA11; PA13; PA14; PA20; PA21; PA23; PB12; PC27; PC60;

M[09P]-8: PA11; PA13; PA14; PA20; PA21; PA23; PA67; PC27; PC60;

M[09P]-9: PA11; PA13; PA14; PA20; PA21; PA23; PA67; PB12; PC60;

M[09P]-10: PA11; PA13; PA14; PA20; PA21; PA23; PA67; PB12; PC27;

M[09P]-21: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12;

M[10P]-41: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20;

M[10P]-303: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33;

M[11P]-1: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA11;

M[11P]-2: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA14;

M[11P]-3: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA25;

M[11P]-4: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA26;

M[11P]-5: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA28;

M[11P]-6: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA29;

M[11P]-7: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA67;

M[11P]-8: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PA75;

M[11P]-9: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49;

M[11P]-10: PA13; PA20; PA21; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC60;

M[11P]-11: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39;

M[11P]-12: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21;

M[12P]-1: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22;

M[12P]-2: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29;

M[13P]-1: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22; PA20;

M[13P]-2: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29; PC20;

M[14P]-1: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22; PA20; PC26;

M[14P]-2: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29; PC20; PC49;

M[15P]-1: PA10; PA11; PA13; PA15; PA20; PA21; PA23; PA26; PA33; PB08; PB12; PB60; PB88; PC12; PC20;

M[15P]-5: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22; PA20; PC26; PC20;

M[16P]-1: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PC11; PC12; PC20; PC51; PC55; PC67;

M[16P]-2: PA11; PA12; PA13; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[16P]-3: PA11; PA12; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[16P]-5: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22; PA20; PC26; PC20; PA12;

M[16P]-6: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29; PC20; PC49; PA14; PA14;

M[17P]-1: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[17P]-2: PA11; PA12; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[17P]-3: PA11; PA12; PA13; PA13; PA13; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[17P]-5: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[17P]-4: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[17P]-6: PA10; PA11; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[17P]-8: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29; PC20; PC49; PA14; PA14; PA22;

M[17P]-9: PA13; PA13; PA13; PC49; PC37; PA11; PA20; PA22; PA12; PC26; PB60; PA39; PC33; PC20; PA14; PA14; PA21;

M[18P]-1: PA10; PA11; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[18P]-2: PA11; PA11; PA12; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-3: PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-4: PA11; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-5: PA11; PA12; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-6: PA11; PA12; PA13; PA13; PA13; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-7: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-9: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-9: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-10: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-11: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-12: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB60; PC20; PC26; PC33; PC37; PC49;

M[18P]-13: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PC20; PC26; PC33; PC37; PC49;

M[18P]-14: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC26; PC33; PC37; PC49;

M[18P]-15: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC33; PC37; PC49;

M[18P]-16: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC37; PC49;

M[18P]-17: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC49;

M[18P]-18: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37;

M[18P]-23: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[18P]-19: PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[18P]-20: PA10; PA10; PA11; PA13; PA15; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[18P]-21: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[18P]-22: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB60; PB88; PC12; PC20;

M[18P]-24: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC12; PC20; PC51; PC55; PC67;

M[18P]-25: PA10; PA11; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[18P]-31: PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[18P]-33: PA11; PA11; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[18P]-34: PA11; PA11; PA13; PA13; PA13; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[18P]-34: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[18P]-35: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22; PA20; PC26; PC20; PA12; PA14; PA14;

M[18P]-36: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29; PC20; PC49; PA14; PA14; PA22; PA20;

M[18P]-37: PA13; PA13; PA13; PC49; PC37; PA11; PA20; PA22; PA12; PC26; PB60; PA39; PC33; PC20; PA14; PA14; PA21; PA29;

M[19P]-1: PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-2: PA11; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-3: PA11; PA13; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-4: PA11; PA13; PA14; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-5: PA11; PA13; PA14; PA20; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-6: PA11; PA13; PA14; PA20; PA21; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-7: PA11; PA13; PA14; PA20; PA21; PA25; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-8: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-9: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-10: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-11: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-12: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-13: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-14: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB33; PB60; PB88; PC20; PC49; PC60;

M[19P]-15: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB60; PB88; PC20; PC49; PC60;

M[19P]-16: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB88; PC20; PC49; PC60;

M[19P]-17: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PC20; PC49; PC60;

M[19P]-18: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC49; PC60;

M[19P]-19: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC60;

M[19P]-20: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49;

M[19P]-21: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-22: PA10; PA11; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-23: PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[19P]-24: PA11; PA11; PA12; PA13; PA13; PA13; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[19P]-25: PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-26: PA10; PA10; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-27: PA10; PA10; PA11; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-28: PA10; PA10; PA11; PA13; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-29: PA10; PA10; PA11; PA13; PA15; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-30: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-31: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-32: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-33: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-34: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB12; PB12; PB60; PB88; PC12; PC20;

M[19P]-35: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB60; PB88; PC12; PC20;

M[19P]-36: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB88; PC12; PC20;

M[19P]-37: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PC12; PC20;

M[19P]-38: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC20;

M[19P]-39: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12;

M[19P]-40: PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-41: PA00; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-42: PA00; PA10; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-44: PA00; PA10; PA20; PA21; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-45: PA00; PA10; PA20; PA21; PA23; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-46: PA00; PA10; PA20; PA21; PA23; PA24; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-47: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-48: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-49: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-50: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC12; PC20; PC51; PC55; PC67;

M[19P]-51: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC20; PC51; PC55; PC67;

M[19P]-52: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC51; PC55; PC67;

M[19P]-53: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC55; PC67;

M[19P]-54: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC67;

M[19P]-55: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55;

M[19P]-56: PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-57: PA10; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-58: PA10; PA11; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-59: PA10; PA11; PA13; PA13; PA13; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-60: PA10; PA11; PA13; PA13; PA13; PA14; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-62: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-62: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-63: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-64: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-65: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-66: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-67: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB33; PB77; PC11; PC52; PC60; PC71;

M[19P]-68: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB77; PC11; PC52; PC60; PC71;

M[19P]-69: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PC11; PC52; PC60; PC71;

M[19P]-70: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC52; PC60; PC71;

M[19P]-71: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC60; PC71;

M[19P]-72: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC71;

M[19P]-73: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60;

M[19P]-89: PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-91: PA11; PA11; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-92: PA11; PA11; PA13; PA13; PA13; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-93: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-93: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-95: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-96: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-96: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[19P]-98: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB28; PB38; PB60; PC37; PC52;

M[19P]-100: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB38; PB60; PC37; PC52;

M[19P]-100: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB60; PC37; PC52;

M[19P]-101: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PC37; PC52;

M[19P]-102: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC52;

M[19P]-103: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37;

M[19P]-104: PC49; PC37; PA13; PA13; PA13; PA11; PA29; PA21; PB12; PC33; PA39; PA22; PA20; PC26; PC20; PA12; PA14; PA14; PB60;

M[19P]-105: PA11; PC37; PC33; PA39; PB60; PC26; PB12; PA13; PA13; PA13; PA21; PA29; PC20; PC49; PA14; PA14; PA22; PA20; PA12;

M[19P]-106: PA13; PA13; PA13; PC49; PC37; PA11; PA20; PA22; PA12; PC26; PB60; PA39; PC33; PC20; PA14; PA14; PA21; PA29; PB12;

M[20P]-29: PA10; PA11; PA12; PA13; PA14; PA15; PA16; PA17; PA18; PA19; PA20; PA21; PA22; PA23; PA24; PA25; PA26; PA27; PA28; PA29;

M[20P]-240: PA21; PA24; PA26; PA33; PA37; PA39; PA67; PB00; PB10; PB12; PB26; PB28; PB33; PB82; PB88; PC20; PC21; PC52; PC60; PC97;

M[20P]-296: PA00; PA11; PA13; PA15; PA21; PA22; PA24; PA26; PA28; PA33; PA39; PA94; PB08; PB12; PB20; PB28; PB33; PB99; PC26; PC52;

M[20P]-297: PA10; PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA29; PA33; PA57; PA67; PA69; PB07; PB12; PB60; PC20; PC37; PC49; PC98;

M[20P]-335: PA00; PA10; PA11; PA21; PA26; PA27; PA31; PA34; PA67; PA69; PA81; PA89; PB01; PB10; PB12; PB25; PB58; PC11; PC86; PC97;

M[20P]-340: PA11; PA13; PA15; PA18; PA21; PA26; PA34; PA36; PA69; PA79; PA95; PB00; PB05; PB07; PB25; PB48; PB60; PC35; PC74; PC84;

M[20P]-349: PA10; PA11; PA13; PA16; PA20; PA33; PA69; PA94; PB07; PB08; PB12; PC01; PC27; PC28; PC49; PC50; PC53; PC55; PC60; PC67;

M[20P]-371: PA05; PA14; PA15; PA21; PA22; PA23; PA25; PA27; PA32; PA34; PA35; PA57; PA72; PA89; PB12; PB39; PB88; PC20; PC55; PC74;

M[20P]-374: PA06; PA13; PA21; PA24; PA25; PA26; PA27; PA57; PB12; PB24; PB28; PB29; PB35; PB38; PB61; PC11; PC37; PC54; PC74; PC86;

M[20P]-375: PA11; PA15; PA19; PA20; PA21; PA22; PA28; PA29; PA67; PA81; PB10; PB12; PB26; PB28; PC23; PC52; PC58; PC60; PC73; PC98;

M[20P]-377: PA07; PA10; PA11; PA13; PA14; PA21; PA22; PA24; PA29; PA32; PA33; PA35; PA57; PB10; PB28; PB36; PB82; PC49; PC52; PC94;

M[20P]-380: PA13; PA15; PA16; PA21; PA24; PA28; PA33; PA38; PA40; PA72; PB12; PB26; PB27; PB82; PC14; PC33; PC55; PC57; PC58; PC98;

M[20P]-382: PA00; PA12; PA13; PA16; PA21; PA24; PA25; PA26; PA28; PA29; PA35; PA57; PA75; PB33; PB88; PC14; PC24; PC36; PC58; PC66;

M[20P]-383: PA10; PA21; PA22; PA23; PA25; PA27; PA81; PA85; PB12; PB26; PB28; PB39; PB77; PB82; PC01; PC17; PC37; PC50; PC52; PC67;

M[20P]-386: PA13; PA14; PA28; PA29; PA33; PA35; PA40; PA57; PA67; PA79; PA88; PB00; PB01; PB07; PB12; PB33; PB77; PB85; PC50; PC66;

M[20P]-387: PA00; PA05; PA10; PA21; PA23; PA24; PA29; PA40; PA94; PA99; PB12; PB82; PB88; PB99; PC11; PC20; PC24; PC34; PC37; PC61;

M[20P]-388: PA12; PA13; PA14; PA15; PA20; PA21; PA26; PA29; PA35; PA40; PA67; PA89; PA98; PB12; PB33; PC01; PC14; PC58; PC68; PC88;

M[20P]-392: PA11; PA14; PA21; PA23; PA26; PA29; PA30; PA57; PA81; PA85; PA94; PB03; PB07; PB12; PB29; PC33; PC51; PC53; PC71; PC73;

M[20P]-396: PA05; PA14; PA15; PA17; PA21; PA28; PA29; PA35; PA37; PA39; PA40; PA69; PA72; PA89; PB10; PB12; PC34; PC67; PC71; PC97;

M[20P]-401: PA13; PA15; PA16; PA23; PA26; PA29; PA33; PA34; PA85; PB12; PB26; PB33; PC21; PC50; PC52; PC53; PC55; PC58; PC67; PC71;

M[20P]-403: PA13; PA14; PA20; PA22; PA24; PA26; PA33; PA35; PB08; PB12; PB26; PB33; PB50; PB61; PC21; PC27; PC37; PC52; PC54; PC58;

M[20P]-407: PA20; PA21; PA22; PA25; PA29; PA48; PA51; PA57; PA67; PA75; PA94; PB07; PB12; PC21; PC34; PC41; PC51; PC53; PC55; PC97;

M[20P]-408: PA11; PA12; PA13; PA15; PA16; PA20; PA21; PA22; PA25; PA26; PA27; PA28; PA29; PB10; PB26; PC20; PC21; PC24; PC25; PC58;

M[20P]-409: PA10; PA15; PA21; PA28; PA35; PA69; PA89; PA94; PA98; PB07; PB12; PB28; PB35; PB36; PC11; PC23; PC49; PC52; PC73; PC98;

M[20P]-416: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[20P]-418: PA10; PA11; PA12; PA13; PA21; PA22; PA23; PA24; PA26; PA39; PB07; PB12; PB38; PC11; PC12; PC20; PC23; PC24; PC49; PC55;

M[20P]-420: PA10; PA14; PA21; PA22; PA23; PA29; PA30; PA33; PA37; PB01; PB12; PB26; PB33; PB99; PC20; PC21; PC36; PC37; PC73; PC98;

M[20P]-421: PA13; PA15; PA21; PA25; PA26; PA29; PA39; PA40; PA75; PA94; PB20; PB55; PB82; PC06; PC20; PC24; PC49; PC71; PC74; PC99;

M[20P]-422: PA10; PA11; PA13; PA18; PA21; PA23; PA25; PA29; PA33; PA57; PA70; PB01; PB07; PB12; PB88; PC11; PC27; PC33; PC55; PC97;

M[20P]-427: PA06; PA13; PA15; PA21; PA24; PA25; PA27; PA29; PA33; PA75; PA98; PB00; PB28; PB60; PC11; PC20; PC26; PC50; PC55; PC74;

M[20P]-429: PA11; PA19; PA21; PA23; PA26; PA33; PA40; PA75; PB00; PB03; PB12; PB60; PC11; PC21; PC24; PC26; PC36; PC37; PC68; PC98;

M[20P]-430: PA00; PA07; PA13; PA23; PA26; PA27; PA34; PA40; PA75; PB07; PB12; PB18; PB28; PB88; PC20; PC41; PC66; PC67; PC71; PC97;

M[20P]-431: PA00; PA06; PA11; PA12; PA13; PA15; PA18; PA21; PA22; PA25; PA28; PA33; PA81; PB09; PB32; PB33; PB38; PC11; PC33; PC52;

M[20P]-432: PA12; PA14; PA19; PA21; PA24; PA26; PA33; PA67; PB05; PB09; PB12; PB28; PB32; PB46; PB60; PC20; PC49; PC58; PC71; PC73;

M[20P]-435: PA10; PA13; PA14; PA15; PA21; PA28; PA33; PA40; PA55; PA57; PB07; PB12; PB58; PB88; PB99; PC12; PC21; PC29; PC66; PC67;

M[20P]-439: PA10; PA13; PA16; PA21; PA24; PA27; PA29; PA34; PA40; PA60; PA75; PB12; PB27; PB35; PB38; PC25; PC26; PC36; PC51; PC55;

M[20P]-443: PA00; PA10; PA13; PA15; PA17; PA20; PA29; PA33; PA35; PA40; PB07; PB12; PB26; PB27; PB82; PC12; PC24; PC50; PC60; PD02;

M[20P]-444: PA11; PA13; PA14; PA18; PA21; PA24; PA28; PA39; PA40; PA57; PA60; PA67; PA88; PB20; PB85; PB88; PC06; PC52; PC57; PC58;

M[20P]-445: PA11; PA13; PA17; PA20; PA21; PA29; PA30; PA33; PA35; PA40; PA57; PB00; PB12; PB28; PB60; PC11; PC20; PC35; PC50; PC51;

M[20P]-446: PA13; PA15; PA16; PA21; PA22; PA23; PA25; PA26; PA27; PA29; PA39; PA67; PA75; PB12; PB26; PB27; PC36; PC49; PC60; PC98;

M[20P]-447: PA11; PA13; PA17; PA19; PA24; PA28; PA30; PA33; PA39; PA40; PA57; PA92; PB07; PB08; PB12; PB33; PB61; PB85; PC58; PC94;

M[20P]-449: PA10; PA11; PA13; PA14; PA15; PA20; PA21; PA24; PA28; PA40; PA67; PA75; PA95; PB12; PB20; PB35; PC14; PC20; PC52; PC60;

M[20P]-453: PA10; PA13; PA15; PA18; PA21; PA25; PA29; PA34; PA35; PA67; PA93; PB06; PB07; PB09; PB12; PB20; PB26; PB35; PB99; PC55;

M[20P]-455: PA00; PA00; PA11; PA11; PA13; PA14; PA18; PA20; PA25; PA28; PA33; PA70; PA75; PB00; PB12; PC20; PC23; PC26; PC67; PC67;

M[20P]-456: PA14; PA21; PA33; PA34; PA39; PB00; PB07; PB07; PB12; PB27; PB28; PB38; PB88; PC20; PC23; PC26; PC49; PC58; PC67; PC71;

M[20P]-457: PA00; PA10; PA11; PA12; PA13; PA13; PA14; PA29; PA29; PA57; PA67; PB02; PB07; PB09; PB12; PB38; PC11; PC60; PC97; PC98;

M[20P]-458: PA10; PA11; PA11; PA13; PA13; PA18; PA21; PA26; PA33; PA57; PA75; PA98; PB12; PB33; PB33; PB38; PB82; PC20; PC55; PC73;

M[20P]-459: PA00; PA06; PA21; PA26; PA26; PA29; PA33; PA75; PB10; PB10; PB12; PB18; PB32; PB85; PC20; PC24; PC49; PC55; PC66; PC98;

M[20P]-461: PA06; PA21; PA29; PA29; PA33; PA33; PA39; PA57; PA67; PB07; PB07; PB12; PB26; PB32; PB32; PB85; PC20; PC27; PC52; PC58;

M[20P]-465: PA11; PA11; PA13; PA16; PA21; PA22; PA27; PA29; PA33; PA69; PB07; PB26; PB28; PB32; PB77; PB88; PB88; PC23; PC60; PC73;

M[20P]-467: PA00; PA06; PA21; PA21; PA23; PA26; PA33; PA33; PA40; PA67; PB12; PB38; PB88; PC11; PC23; PC23; PC27; PC55; PC71; PC97;

M[20P]-469: PA00; PA12; PA13; PA13; PA13; PA21; PA29; PA33; PA33; PA34; PA57; PB07; PC11; PC11; PC20; PC23; PC23; PC55; PC74; PC97;

M[20P]-470: PA13; PA14; PA21; PA21; PA22; PA23; PA24; PA24; PA27; PA29; PA33; PA67; PA75; PB07; PB07; PB33; PB33; PB77; PC37; PC52;

M[20P]-472: PA10; PA10; PA11; PA13; PA21; PA25; PA25; PA28; PA33; PA39; PB07; PB12; PB88; PC12; PC12; PC20; PC20; PC24; PC55; PC71;

M[20P]-474: PA00; PA13; PA20; PA21; PA26; PA28; PA29; PA33; PA57; PA67; PA81; PA81; PA98; PB02; PB12; PB38; PB60; PB88; PC11; PC20;

M[20P]-475: PA11; PA12; PA12; PA13; PA28; PA28; PA29; PA29; PA32; PA52; PA52; PA75; PA81; PB12; PB20; PB33; PB60; PC21; PC49; PC51;

M[20P]-478: PA15; PA16; PA21; PA21; PA21; PA23; PA25; PA32; PA34; PA39; PB12; PB77; PB85; PB88; PB88; PB88; PB88; PC11; PC33; PC37;

M[20P]-479: PA10; PA11; PA13; PA13; PA18; PA20; PA21; PA28; PA32; PA40; PA67; PA69; PB07; PB07; PB10; PB12; PB20; PC55; PC60; PC98;

M[20P]-480: PA00; PA06; PA13; PA22; PA23; PA26; PA26; PA27; PA29; PA40; PA81; PB07; PB10; PB10; PB12; PB12; PB18; PB32; PC49; PC71;

M[20P]-481: PA11; PA13; PA26; PA29; PA29; PA35; PA69; PA75; PA75; PB02; PB12; PB33; PC20; PC23; PC23; PC37; PC41; PC50; PC52; PC98;

M[20P]-482: PA06; PA10; PA11; PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA20; PA25; PA26; PA28; PA39; PA67; PB12; PC11; PC24; PC74;

M[20P]-484: PA11; PA13; PA23; PA28; PA29; PA29; PA35; PA39; PA40; PA60; PA75; PA98; PB02; PB12; PB38; PB38; PB82; PC20; PC35; PC50;

M[20P]-485: PA13; PA14; PA14; PA21; PA21; PA21; PA26; PA26; PA26; PA26; PA29; PA29; PA52; PA57; PB00; PC11; PC11; PC21; PC51; PC67;

M[20P]-486: PA11; PA13; PA23; PA24; PA35; PA35; PA35; PA69; PA69; PA75; PB07; PB12; PB27; PC20; PC20; PC21; PC23; PC50; PC55; PC73;

M[20P]-490: PA10; PA11; PA11; PA14; PA21; PA24; PA26; PA26; PA39; PA40; PA69; PA75; PB00; PB01; PB12; PB12; PB18; PB28; PB60; PC20;

M[20P]-493: PA06; PA14; PA20; PA21; PA21; PA28; PA29; PA33; PA69; PA81; PA98; PB12; PB38; PB60; PC11; PC12; PC49; PC55; PC71; PD02;

M[20P]-494: PA06; PA10; PA11; PA11; PA13; PA13; PA15; PA23; PA26; PA35; PB01; PB12; PB60; PC12; PC20; PC37; PC41; PC49; PC49; PC52;

M[20P]-495: PA10; PA10; PA13; PA14; PA18; PA20; PA21; PA26; PA29; PA29; PA57; PB02; PC11; PC11; PC20; PC20; PC24; PC33; PC49; PC49;

M[20P]-496: PA00; PA18; PA21; PA24; PA26; PA27; PA29; PA39; PA39; PA52; PA69; PA70; PA98; PB00; PB02; PB12; PC12; PC24; PC55; PC67;

M[20P]-497: PA11; PA11; PA13; PA13; PA15; PA21; PA23; PA24; PA25; PA26; PA28; PA39; PA52; PA67; PB00; PB12; PB27; PB28; PC12; PC37;

M[20P]-499: PA13; PA21; PA21; PA21; PA29; PA30; PA69; PA75; PB07; PB07; PB07; PB60; PC20; PC20; PC21; PC37; PC37; PC49; PC55; PD02;

M[20P]-503: PA06; PA06; PA10; PA10; PA13; PA13; PA20; PA21; PA25; PA26; PA29; PA33; PA39; PA70; PB28; PC11; PC20; PC23; PC49; PC97;

M[20P]-506: PA11; PA16; PA21; PA23; PA29; PA39; PA57; PB12; PB27; PB32; PB88; PB88; PB88; PC12; PC20; PC21; PC37; PC71; PD02; PD02;

M[20P]-510: PA13; PA21; PA25; PA26; PA27; PA33; PA39; PA40; PA69; PA81; PB01; PB28; PB60; PB85; PB88; PC27; PC41; PC41; PC50; PC55;

M[20P]-514: PA11; PA12; PA13; PA13; PA21; PA21; PA28; PA29; PA33; PA35; PA39; PA40; PA40; PA67; PA67; PB12; PC14; PC20; PC41; PC55;

M[20P]-516: PA07; PA10; PA13; PA14; PA17; PA21; PA28; PA29; PA39; PA40; PA40; PA69; PB07; PB12; PC21; PC21; PC27; PC37; PD02; PD02;

M[20P]-520: PA11; PA11; PA12; PA13; PA14; PA15; PA29; PA33; PA33; PA57; PA67; PA70; PA98; PB12; PC20; PC23; PC33; PC52; PC55; PD02;

M[20P]-523: PA10; PA11; PA18; PA20; PA21; PA33; PA39; PA67; PA67; PA69; PB07; PB12; PB28; PB88; PB88; PC21; PC33; PC35; PC49; PC50;

M[20P]-524: PA00; PA11; PA11; PA12; PA13; PA20; PA29; PA40; PB07; PB12; PB28; PB38; PB60; PB60; PB77; PC20; PC20; PC49; PC49; PC98;

M[20P]-525: PA11; PA11; PA13; PA15; PA21; PA26; PA26; PA27; PA28; PA29; PA29; PA30; PA40; PA52; PA75; PB07; PB07; PC11; PC33; PC37;

M[20P]-527: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26; PA26; PA26; PA33; PB08; PB12; PB12; PB60; PB88; PC12; PC20;

M[20P]-532: PA14; PA20; PA20; PA21; PA22; PA25; PA26; PA27; PA28; PA29; PA33; PA40; PA75; PB00; PB12; PB12; PB60; PB85; PC12; PC51;

M[20P]-533: PA13; PA21; PA26; PA27; PA27; PA28; PA32; PA60; PA70; PA75; PB18; PB32; PB38; PC23; PC41; PC50; PC50; PC51; PC52; PC97;

M[20P]-535: PA11; PA21; PA26; PA28; PA29; PA29; PA29; PA34; PA57; PB07; PB12; PB12; PB12; PB33; PB60; PC20; PC20; PC51; PC55; PC67;

M[20P]-536: PA00; PA10; PA11; PA12; PA12; PA13; PA14; PA23; PA29; PA29; PA35; PA40; PA81; PB07; PB07; PB12; PB27; PB33; PB60; PC20;

M[20P]-538: PA21; PA23; PA25; PA26; PA27; PA28; PA75; PB07; PB12; PB27; PB32; PB88; PC11; PC11; PC12; PC20; PC33; PC49; PC55; PC97;

M[20P]-541: PA10; PA11; PA13; PA20; PA24; PA28; PA29; PA67; PA70; PB10; PB12; PB77; PC20; PC20; PC23; PC41; PC51; PC52; PC52; PC97;

M[20P]-548: PA10; PA13; PA14; PA14; PA22; PA27; PA27; PA28; PA29; PA29; PA33; PA39; PB10; PB12; PB60; PB88; PC20; PC37; PC50; PC55;

M[20P]-551: PA12; PA13; PA13; PA20; PA22; PA23; PA24; PA24; PA26; PA33; PA33; PA39; PA67; PB07; PB07; PB12; PB33; PC20; PC55; PC98;

M[20P]-552: PA06; PA11; PA11; PA13; PA14; PA14; PA20; PA21; PA21; PA21; PA24; PA35; PB00; PB02; PB12; PB60; PB60; PC12; PC21; PC98;

M[20P]-555: PA00; PA10; PA11; PA18; PA20; PA20; PA21; PA25; PA26; PA26; PA39; PA39; PA39; PA89; PB12; PB12; PC20; PC20; PC49; PC74;

M[20P]-557: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12; PB12; PB12; PB12; PC11; PC11; PC12; PC20; PC51; PC55; PC67;

M[20P]-559: PA00; PA11; PA12; PA13; PA20; PA22; PA24; PA25; PA25; PA26; PA28; PA57; PA70; PA70; PB07; PB12; PB88; PB88; PC11; PC60;

M[20P]-560: PA10; PA10; PA11; PA12; PA13; PA14; PA20; PA25; PA28; PA28; PA29; PA67; PB12; PB38; PB60; PC24; PC35; PC41; PC51; PC97;

M[20P]-563: PA12; PA13; PA20; PA20; PA21; PA24; PA25; PA25; PA29; PA29; PA33; PA81; PB33; PB60; PB60; PB88; PB88; PB88; PC11; PC41;

M[20P]-564: PA07; PA13; PA13; PA21; PA26; PA37; PA39; PA67; PA67; PB02; PB07; PB07; PB12; PB12; PB28; PC12; PC37; PC97; PC97; PC97;

M[20P]-565: PA07; PA11; PA13; PA20; PA29; PA29; PA32; PA75; PA95; PA98; PB02; PB12; PB32; PB33; PB60; PB85; PC20; PC24; PC35; PC41;

M[20P]-567: PA10; PA13; PA14; PA16; PA21; PA26; PA28; PA75; PA81; PB07; PB07; PB12; PB88; PC23; PC24; PC24; PC33; PC52; PC55; PC67;

M[20P]-569: PA13; PA14; PA21; PA26; PA27; PA39; PA57; PA67; PB02; PB07; PB12; PB28; PC12; PC21; PC49; PC55; PC55; PC67; PC67; PC98;

M[20P]-572: PA13; PA14; PA21; PA27; PA29; PA33; PA67; PA69; PA79; PB00; PB01; PB12; PB33; PB38; PC37; PC41; PC49; PC51; PC55; PD02;

M[20P]-573: PA10; PA11; PA13; PA13; PA13; PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[20P]-574: PA13; PA21; PA22; PA23; PA25; PA29; PA35; PA67; PB01; PB12; PB12; PB35; PB88; PC24; PC37; PC37; PC51; PC52; PC52; PC66;

M[20P]-575: PA11; PA11; PA13; PA13; PA13; PA14; PA14; PA26; PA26; PA28; PA52; PA67; PB00; PB12; PB27; PB28; PB38; PB60; PC37; PC52;

M[20P]-576: PA11; PA11; PA12; PA13; PA13; PA13; PA14; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[20P]-577: PA06; PA13; PA13; PA14; PA22; PA22; PA23; PA26; PA28; PA69; PA70; PB07; PB12; PB12; PB32; PC12; PC20; PC24; PC24; PC98;

M[20P]-579: PA12; PA13; PA13; PA13; PA14; PA26; PA26; PA28; PA57; PA81; PB02; PB07; PB12; PC11; PC12; PC20; PC51; PC55; PD02; PD02;

M[20P]-580: PA00; PA11; PA12; PA13; PA20; PA21; PA21; PA25; PA26; PA26; PA33; PA75; PA75; PA75; PB28; PB88; PC20; PC76; PC98; PC98;

M[20P]-581: PA10; PA11; PA13; PA21; PA22; PA25; PA29; PA67; PB07; PB60; PB88; PB88; PC20; PC21; PC24; PC37; PC41; PC55; PC66; PC98;

M[20P]-582: PA10; PA11; PA12; PA13; PA20; PA21; PA23; PA25; PA39; PA75; PB07; PB07; PB38; PB38; PB60; PB60; PC20; PC24; PC41; PC66;

M[20P]-583: PA11; PA13; PA14; PA23; PA25; PA25; PA26; PA26; PA28; PA67; PB12; PB18; PB88; PC24; PC24; PC37; PC41; PC49; PC55; PC60;

M[20P]-584: PA00; PA14; PA21; PA26; PA26; PA28; PA39; PA39; PA67; PA75; PB12; PB12; PB27; PB33; PB60; PC11; PC41; PC49; PC55; PC97;

M[20P]-588: PA07; PA13; PA14; PA21; PA26; PA26; PA26; PA26; PA32; PA67; PA67; PA75; PB12; PB27; PB33; PB60; PC20; PC20; PC23; PC24;

M[20P]-589: PA13; PA13; PA22; PA25; PA25; PA27; PA29; PA29; PA35; PA39; PA52; PA75; PB12; PB32; PB38; PB85; PB88; PC11; PC23; PC98;

M[20P]-590: PA11; PA13; PA13; PA14; PA25; PA29; PA33; PA35; PA35; PA35; PA39; PB02; PB12; PC11; PC20; PC20; PC21; PC27; PC55; PC55;

M[20P]-592: PA10; PA10; PA11; PA11; PA13; PA14; PA21; PA21; PA26; PA26; PA27; PA29; PA30; PA75; PB07; PB07; PB12; PB38; PC20; PC24;

M[20P]-594: PA13; PA21; PA21; PA21; PA23; PA27; PA28; PA39; PA60; PA69; PA70; PB02; PB09; PB12; PB12; PB88; PC24; PC49; PC97; PC98;

M[20P]-595: PA11; PA13; PA22; PA23; PA25; PA28; PA28; PA28; PA29; PA29; PA39; PA67; PA69; PB12; PB60; PC12; PC20; PC24; PC49; PC55;

M[20P]-596: PA11; PA11; PA13; PA13; PA14; PA21; PA22; PA23; PA26; PA26; PA67; PB12; PB60; PC11; PC20; PC20; PC37; PC50; PC55; PC60;

M[20P]-599: PA13; PA13; PA14; PA16; PA26; PA29; PA75; PA75; PB07; PB12; PB60; PB60; PB60; PB60; PC12; PC20; PC20; PC37; PC37; PC52;

M[20P]-601: PA11; PA11; PA13; PA13; PA13; PA23; PA26; PA29; PA29; PA35; PB02; PB10; PB12; PB33; PC20; PC21; PC41; PC49; PC60; PC67;

M[20P]-602: PA00; PA10; PA10; PA14; PA14; PA14; PA21; PA25; PA26; PA29; PA29; PB07; PB07; PB07; PB12; PB12; PC20; PC52; PC55; PD02;

M[20P]-603: PA11; PA13; PA16; PA20; PA21; PA23; PA23; PA26; PA26; PA28; PA33; PA39; PA39; PA67; PB60; PB60; PC20; PC23; PC49; PC55;

M[20P]-604: PA10; PA13; PA14; PA14; PA18; PA21; PA22; PA23; PA26; PA28; PA28; PA29; PB12; PB33; PC11; PC11; PC51; PC51; PC60; PC71;

M[20P]-605: PA11; PA11; PA11; PA11; PA13; PA13; PA13; PA20; PA25; PA29; PA39; PA39; PA69; PA81; PB07; PB12; PB88; PC20; PC41; PC55;

M[20P]-607: PA10; PA11; PA11; PA13; PA13; PA13; PA22; PA22; PA26; PA33; PA35; PA75; PA75; PA81; PB12; PB88; PC20; PC49; PC55; PC97;

M[20P]-608: PA00; PA13; PA13; PA13; PA18; PA26; PA27; PA29; PA35; PA57; PA69; PA75; PA98; PB12; PB60; PC11; PC20; PC20; PC55; PC60;

M[20P]-609: PA11; PA14; PA21; PA22; PA23; PA26; PA34; PA52; PA67; PA69; PA69; PB07; PB12; PB12; PB27; PB77; PC12; PC20; PC55; PC97;

M[20P]-612: PA11; PA13; PA14; PA14; PA14; PA21; PA22; PA23; PA52; PA81; PB00; PB07; PB12; PB33; PB38; PB60; PC12; PC24; PC33; PC37;

M[20P]-613: PA10; PA13; PA13; PA14; PA21; PA21; PA22; PA23; PA26; PA28; PA28; PA29; PB12; PB12; PB27; PB60; PC20; PC21; PC21; PC60;

M[20P]-616: PA13; PA26; PA28; PA29; PA39; PA57; PA67; PB07; PB12; PB38; PB60; PB77; PB77; PB88; PC33; PC49; PC52; PC52; PC98; PC98;

M[20P]-618: PA11; PA12; PA13; PA13; PA14; PA20; PA21; PA22; PA23; PA26; PA29; PA67; PA79; PB88; PC11; PC20; PC20; PC37; PC41; PC55;

M[20P]-620: PA12; PA13; PA20; PA22; PA22; PA23; PA26; PA26; PA28; PA39; PA69; PA95; PB12; PB60; PB77; PC20; PC21; PC52; PC60; PC98;

M[20P]-621: PA10; PA13; PA14; PA24; PA24; PA26; PA27; PA29; PA32; PA35; PA67; PB12; PB12; PB12; PB27; PB33; PB33; PB38; PB38; PB38;

M[20P]-622: PA10; PA12; PA14; PA14; PA15; PA20; PA21; PA26; PA27; PA32; PA40; PB10; PB12; PB12; PB88; PC20; PC20; PC21; PC52; PC60;

M[20P]-623: PA10; PA11; PA13; PA14; PA20; PA24; PA25; PA26; PA26; PA28; PA39; PA81; PB09; PB12; PB28; PB88; PC24; PC41; PC52; PC98;

M[20P]-626: PA11; PA11; PA13; PA13; PA20; PA21; PA21; PA25; PA27; PA27; PA28; PA39; PA39; PA40; PA57; PB12; PB60; PC20; PC37; PC41;

M[20P]-631: PA10; PA13; PA20; PA21; PA21; PA23; PA23; PA26; PA29; PA39; PA39; PA67; PB10; PB38; PB60; PC11; PC20; PC37; PC49; PC66;

M[20P]-632: PA10; PA11; PA18; PA21; PA22; PA23; PA28; PA29; PA33; PA34; PA69; PB07; PB12; PB60; PC20; PC20; PC20; PC37; PC41; PC55;

M[20P]-633: PA10; PA11; PA11; PA13; PA20; PA34; PA39; PB00; PB07; PB12; PB12; PB60; PB88; PB88; PC11; PC12; PC23; PC35; PC49; PC52;

M[20P]-634: PA13; PA13; PA13; PA21; PA22; PA22; PA25; PA25; PA28; PA28; PA29; PA67; PA81; PB00; PB12; PB12; PB27; PB33; PC11; PC98;

M[20P]-635: PA00; PA14; PA17; PA20; PA21; PA23; PA26; PA27; PA75; PA81; PB00; PB12; PB82; PC11; PC20; PC26; PC51; PC52; PC55; PC71;

M[20P]-638: PA00; PA14; PA17; PA21; PA22; PA24; PA28; PA33; PA52; PA57; PA75; PB00; PB07; PB12; PB28; PB88; PC20; PC37; PC41; PC97;

M[20P]-649: PA06; PA10; PA11; PA12; PA13; PA15; PA16; PA20; PA24; PA30; PA33; PA98; PB10; PB12; PB28; PB32; PB60; PC11; PC20; PC66;

M[20P]-654: PA00; PA11; PA15; PA20; PA21; PA26; PA32; PA34; PA35; PA39; PA57; PB10; PB12; PB18; PC11; PC20; PC21; PC50; PC55; PC66;

M[20P]-655: PA11; PA13; PA14; PA20; PA22; PA26; PA28; PA30; PA40; PA52; PA57; PA81; PB12; PB32; PB85; PC41; PC50; PC55; PC66; PC71;

M[20P]-658: PA10; PA11; PA12; PA13; PA14; PA15; PA17; PA20; PA21; PA28; PA29; PA52; PA67; PB10; PB12; PB18; PB60; PC20; PC41; PC66;

M[20P]-659: PA00; PA11; PA21; PA22; PA24; PA26; PA27; PA28; PA29; PA33; PA35; PA81; PB07; PB12; PB18; PB28; PB85; PC37; PC49; PC74;

M[20P]-663: PA06; PA13; PA20; PA25; PA26; PA27; PA28; PA40; PA67; PA81; PB00; PB12; PB18; PB33; PB61; PC20; PC24; PC37; PC41; PC52;

M[20P]-666: PA06; PA13; PA21; PA23; PA27; PA28; PA30; PA40; PA75; PB00; PB07; PB10; PB12; PB18; PB28; PB33; PC20; PC49; PC52; PC71;

M[20P]-668: PA10; PA16; PA17; PA21; PA24; PA27; PA28; PA33; PA40; PA57; PA75; PA79; PB07; PB08; PB12; PB33; PB88; PC20; PC35; PC97;

M[20P]-670: PA11; PA12; PA13; PA14; PA20; PA23; PA27; PA28; PA35; PA39; PA67; PA79; PA81; PA89; PB10; PB12; PB18; PC20; PC21; PC51;

M[20P]-678: PA06; PA11; PA12; PA13; PA21; PA23; PA29; PA35; PA39; PA40; PB00; PB07; PB10; PB12; PB33; PB60; PB77; PC11; PC67; PC76;

M[20P]-681: PA07; PA10; PA11; PA21; PA24; PA26; PA30; PA35; PA39; PA57; PA75; PB07; PB12; PB28; PB88; PC11; PC49; PC55; PC58; PC66;

M[20P]-686: PA00; PA10; PA13; PA22; PA24; PA27; PA28; PA33; PA39; PA40; PA69; PB12; PB32; PB38; PB85; PB88; PC20; PC55; PC60; PC66;

M[20P]-692: PA07; PA10; PA13; PA21; PA22; PA26; PA34; PA39; PA40; PA81; PB12; PB18; PB32; PB33; PB88; PC35; PC37; PC52; PC55; PC76;

M[20P]-694: PA07; PA11; PA13; PA14; PA15; PA16; PA21; PA27; PA28; PA29; PA35; PA39; PA40; PA75; PB12; PB33; PB88; PC50; PC51; PC52;

M[20P]-697: PA00; PA13; PA16; PA19; PA21; PA22; PA25; PA27; PA28; PA39; PA79; PA81; PB00; PB12; PB32; PB60; PB99; PC51; PC58; PC76;

M[20P]-703: PA00; PA13; PA14; PA15; PA24; PA26; PA33; PA35; PA52; PA75; PA81; PB07; PB12; PB28; PB85; PC20; PC21; PC35; PC41; PD02;

M[20P]-705: PA11; PA13; PA30; PA33; PA40; PA67; PA75; PB07; PB12; PB28; PB35; PB88; PC20; PC21; PC24; PC35; PC37; PC41; PC50; PC55;

M[20P]-708: PA07; PA13; PA23; PA25; PA27; PA29; PA30; PA33; PA52; PA57; PA75; PB00; PB08; PB12; PB38; PB60; PB77; PC50; PC76; PD02;

M[20P]-709: PA11; PA13; PA17; PA20; PA21; PA23; PA26; PA27; PA28; PA40; PA69; PA81; PA92; PB00; PB07; PB12; PC20; PC41; PC52; PC66;

M[20P]-712: PA12; PA13; PA15; PA20; PA22; PA26; PA27; PA29; PA35; PA52; PA81; PB07; PB10; PB12; PB33; PB88; PC11; PC20; PC41; PC52;

M[20P]-714: PA07; PA10; PA11; PA16; PA21; PA26; PA33; PA40; PA67; PA81; PA98; PB00; PB07; PB12; PB28; PC11; PC41; PC49; PC50; PC98;

M[20P]-716: PA11; PA13; PA15; PA19; PA22; PA26; PA33; PA37; PA39; PA57; PB07; PB12; PB28; PB33; PB88; PB99; PC14; PC23; PC35; PC67;

M[20P]-718: PA12; PA13; PA15; PA16; PA20; PA22; PA25; PA26; PA39; PA40; PA92; PB07; PB12; PB27; PB35; PB38; PB99; PC11; PC27; PC41;

M[20P]-721: PA19; PA20; PA21; PA24; PA25; PA27; PA29; PA30; PA55; PA57; PB12; PB32; PB58; PB60; PB88; PB99; PC41; PC50; PC60; PC67;

M[20P]-723: PA12; PA13; PA16; PA17; PA21; PA28; PA29; PA33; PA34; PA40; PA60; PA89; PB28; PB60; PB61; PC14; PC24; PC41; PC51; PC74;

M[20P]-724: PA13; PA23; PA25; PA35; PA57; PA67; PB07; PB12; PB32; PB33; PB88; PB99; PC12; PC20; PC23; PC49; PC50; PC66; PC67; PC94;

M[20P]-725: PA00; PA13; PA14; PA20; PA22; PA25; PA26; PA28; PA33; PA40; PA57; PA81; PB12; PB16; PB33; PB88; PC11; PC24; PC51; PC74;

M[20P]-733: PA11; PA13; PA15; PA21; PA28; PA30; PA33; PA40; PB12; PB32; PB33; PB99; PC11; PC12; PC20; PC60; PC73; PC74; PC94; PD02;

M[20P]-737: PA00; PA11; PA13; PA15; PA17; PA21; PA24; PA28; PA29; PA30; PA67; PB07; PB27; PB28; PB77; PC20; PC21; PC41; PC51; PC94;

M[20P]-740: PA11; PA13; PA14; PA15; PA22; PA23; PA28; PA33; PA39; PA57; PA75; PA89; PB02; PB12; PB28; PB33; PC12; PC21; PC24; PC55;

M[20P]-745: PA11; PA21; PA22; PA24; PA26; PA27; PA28; PA29; PA57; PA89; PB07; PB12; PB28; PB33; PC08; PC11; PC41; PC66; PC67; PC98;

M[20P]-746: PA20; PA21; PA25; PA26; PA28; PA33; PA39; PA40; PA81; PB06; PB07; PB12; PB32; PB60; PC20; PC21; PC24; PC37; PC50; PC55;

M[20P]-748: PA11; PA13; PA14; PA17; PA23; PA26; PA32; PA57; PA60; PA81; PB07; PB12; PB28; PB32; PB33; PB85; PB99; PC20; PC21; PC35;

M[20P]-752: PA11; PA13; PA18; PA26; PA28; PA29; PA33; PA35; PA39; PA67; PA75; PB07; PB12; PB32; PB88; PB99; PC12; PC50; PC54; PC62;

M[20P]-753: PA11; PA12; PA13; PA14; PA15; PA29; PA32; PA40; PA57; PA81; PB00; PB12; PB33; PB60; PC20; PC21; PC49; PC52; PC66; PD02;

M[20P]-757: PA11; PA13; PA21; PA22; PA23; PA27; PA34; PA35; PA40; PA57; PA67; PB10; PB12; PB22; PB58; PC11; PC14; PC23; PC55; PC98;

M[20P]-758: PA10; PA11; PA13; PA20; PA21; PA23; PA28; PA29; PA30; PA40; PA69; PA75; PA81; PB12; PC06; PC20; PC41; PC52; PC57; PC74;

M[20P]-764: PA11; PA13; PA22; PA29; PA35; PA40; PA57; PA73; PA75; PA81; PB08; PB12; PB33; PB82; PC11; PC20; PC21; PC29; PC41; PC52;

M[20P]-765: PA10; PA11; PA13; PA15; PA26; PA27; PA30; PA35; PA40; PA89; PB12; PB22; PB58; PB60; PC34; PC37; PC55; PC58; PC67; PD02;

M[20P]-766: PA11; PA13; PA17; PA22; PA24; PA25; PA29; PA30; PA33; PA34; PA35; PA81; PA92; PB00; PB12; PB82; PC24; PC50; PC74; PC97;

M[20P]-767: PA11; PA21; PA22; PA28; PA29; PA33; PA37; PB12; PB60; PB85; PB88; PB99; PC14; PC20; PC21; PC41; PC51; PC55; PC57; PC98;

M[20P]-771: PA10; PA11; PA13; PA14; PA22; PA26; PA27; PA28; PA29; PA35; PA86; PA89; PB07; PB12; PB28; PB88; PB99; PC12; PC21; PC50;

M[20P]-777: PA11; PA14; PA15; PA20; PA21; PA24; PA25; PA28; PA29; PA33; PA75; PB12; PB85; PB99; PC21; PC51; PC55; PC62; PC67; PD02;

M[20P]-779: PA15; PA19; PA20; PA21; PA22; PA23; PA24; PA25; PA29; PA30; PA57; PA67; PA75; PA89; PB07; PB12; PB32; PC20; PC51; PC74;

M[20P]-781: PA10; PA15; PA21; PA22; PA39; PA57; PA75; PB08; PB09; PB12; PB28; PB35; PB58; PB60; PC12; PC33; PC37; PC66; PC67; PC74;

M[20P]-782: PA10; PA11; PA12; PA13; PA14; PA20; PA26; PA30; PA75; PA89; PA92; PB05; PB07; PB12; PB32; PB82; PB99; PC51; PC58; PC73;

M[20P]-785: PA16; PA17; PA21; PA28; PA29; PA30; PA32; PA33; PA55; PB07; PB12; PB77; PB85; PB88; PB99; PC12; PC20; PC35; PC67; PC98;

M[20P]-786: PA11; PA13; PA14; PA18; PA19; PA21; PA22; PA27; PA28; PA29; PA33; PA40; PA52; PA57; PA81; PB28; PC11; PC20; PC21; PC50;

M[20P]-787: PA10; PA11; PA13; PA22; PA23; PA35; PB07; PB12; PB22; PB85; PB99; PC11; PC20; PC41; PC49; PC52; PC53; PC55; PC73; PD02;

M[20P]-789: PA10; PA11; PA13; PA22; PA23; PA27; PA40; PA57; PA75; PA81; PB07; PB12; PB27; PB60; PB88; PC11; PC20; PC23; PC35; PC41;

M[20P]-791: PA10; PA11; PA13; PA15; PA21; PA33; PA35; PA40; PA57; PA69; PA85; PA98; PA99; PB07; PB12; PB20; PB99; PC12; PC20; PC58;

M[20P]-793: PA11; PA13; PA14; PA15; PA19; PA26; PA29; PA55; PA67; PA89; PB12; PB33; PB88; PC14; PC20; PC21; PC34; PC37; PC67; PC97;

M[20P]-794: PA21; PA23; PA28; PA29; PA34; PA35; PA57; PA75; PA81; PB07; PB12; PB18; PB32; PB58; PC11; PC14; PC41; PC55; PC62; PC74;

M[20P]-795: PA10; PA13; PA20; PA23; PA27; PA29; PA57; PA60; PA69; PB00; PB07; PB12; PB18; PB32; PB85; PB88; PC20; PC66; PC74; PD02;

M[20P]-803: PA11; PA13; PA15; PA22; PA25; PA26; PA29; PA30; PA33; PA40; PA75; PB07; PB12; PB28; PC21; PC23; PC34; PC55; PC71; PC73;

M[20P]-804: PA11; PA12; PA13; PA20; PA22; PA23; PA30; PA33; PA34; PA79; PB12; PB18; PB58; PB60; PB85; PB88; PC20; PC50; PC67; PC68;

M[20P]-806: PA06; PA10; PA11; PA13; PA19; PA20; PA21; PA23; PA25; PA28; PA52; PA73; PA75; PA98; PB12; PB33; PB82; PB85; PB88; PC11;

M[20P]-816: PA11; PA13; PA19; PA25; PA28; PA29; PA33; PA57; PA81; PA89; PB00; PB10; PB12; PB26; PB28; PB99; PC11; PC12; PC76; PC97;

M[20P]-819: PA10; PA11; PA13; PA15; PA26; PA34; PA60; PA81; PA92; PB07; PB12; PB38; PB85; PC01; PC14; PC20; PC52; PC58; PC66; PC67;

M[20P]-823: PA00; PA11; PA12; PA13; PA14; PA17; PA27; PA29; PA40; PA60; PA75; PA79; PA89; PB07; PB12; PB99; PC20; PC52; PC66; PC71;

M[20P]-830: PA13; PA20; PA25; PA26; PA28; PA40; PA75; PA89; PB00; PB07; PB12; PB32; PB88; PC06; PC12; PC20; PC41; PC52; PC76; PC98;

M[20P]-831: PA11; PA15; PA21; PA25; PA33; PB12; PB28; PB32; PB33; PB38; PB60; PB77; PB88; PC20; PC23; PC35; PC37; PC49; PC58; PC66;

M[20P]-841: PA10; PA11; PA13; PA18; PA22; PA28; PA29; PA30; PA35; PA40; PA55; PA75; PB07; PB12; PB28; PB32; PB33; PC41; PC49; PC60;

M[20P]-844: PA11; PA15; PA21; PA24; PA26; PA29; PA33; PA40; PA69; PA81; PA98; PB12; PB28; PB61; PC49; PC67; PC71; PC73; PC74; PD02;

M[20P]-845: PA11; PA13; PA14; PA15; PA21; PA30; PA34; PA39; PA60; PA79; PA81; PA89; PB07; PB18; PC14; PC20; PC21; PC34; PC68; PC97;

M[20P]-846: PA11; PA13; PA25; PA27; PA29; PA32; PA33; PA35; PA37; PA39; PA40; PB08; PB12; PB33; PB88; PC24; PC41; PC52; PC58; PC62;

M[20P]-847: PA10; PA19; PA20; PA21; PA28; PA29; PA30; PA67; PA75; PA89; PB00; PB12; PB28; PB33; PB60; PC12; PC55; PC73; PC74; PD02;

M[20P]-848: PA07; PA11; PA13; PA15; PA20; PA22; PA26; PA27; PA30; PA40; PA69; PA75; PB12; PB32; PB85; PB88; PC34; PC37; PC66; PC68;

M[20P]-849: PA00; PA13; PA25; PA27; PA28; PA29; PA34; PA40; PA57; PA81; PB00; PB12; PB18; PB77; PC20; PC24; PC35; PC41; PC50; PC51;

M[20P]-852: PA00; PA11; PA13; PA15; PA19; PA22; PA26; PA35; PA89; PB00; PB12; PB58; PB77; PC12; PC20; PC29; PC62; PC71; PC94; PC98;

M[20P]-853: PA00; PA11; PA21; PA24; PA25; PA33; PA57; PA60; PB07; PB12; PB28; PB32; PB60; PB82; PB88; PC24; PC41; PC50; PC51; PC58;

M[20P]-857: PA00; PA11; PA13; PA26; PA28; PA75; PA76; PA79; PB12; PB28; PB60; PB88; PC11; PC12; PC20; PC37; PC50; PC52; PC67; PD02;

M[20P]-873: PA10; PA13; PA26; PA27; PA30; PA34; PA85; PA98; PB05; PB07; PB12; PB28; PB29; PB32; PB60; PC14; PC20; PC26; PC41; PC76;

M[20P]-880: PA11; PA13; PA17; PA20; PA22; PA24; PA26; PA27; PA29; PA38; PA57; PB07; PB12; PB20; PB29; PB97; PC24; PC37; PC51; PC71;

M[20P]-888: PA10; PA12; PA15; PA16; PA21; PA27; PA28; PA33; PA44; PB00; PB12; PB13; PB27; PC11; PC20; PC51; PC58; PC73; PC75; PD02;

M[20P]-890: PA00; PA11; PA13; PA15; PA16; PA25; PA34; PA75; PA82; PA86; PB00; PB12; PB16; PB27; PB33; PC20; PC49; PC50; PC57; PC98;

M[20P]-894: PA12; PA18; PA21; PA24; PA26; PA27; PA34; PA57; PA67; PA82; PB01; PB12; PB33; PB76; PB98; PB99; PC11; PC24; PC33; PC83;

M[20P]-902: PA12; PA13; PA21; PA26; PA29; PA39; PA40; PA57; PA96; PB00; PB07; PB12; PB27; PC20; PC50; PC53; PC61; PC62; PC75; PC94;

M[20P]-904: PA06; PA13; PA20; PA23; PA25; PA27; PA35; PA46; PA52; PA60; PA81; PA90; PA97; PB03; PB12; PB57; PB99; PC20; PC52; PC73;

M[20P]-907: PA00; PA07; PA14; PA21; PA24; PA25; PA45; PA75; PA83; PA97; PB12; PB26; PB32; PB60; PB92; PC06; PC20; PC35; PC37; PD02;

M[20P]-909: PA12; PA14; PA20; PA21; PA29; PA39; PA72; PA93; PB12; PB22; PB28; PB32; PC21; PC23; PC24; PC41; PC52; PC73; PC79; PD02.

Note: if in the previous and in the following analogous Lists a plant code occurs more than one time in the same herbal composition, it means that more than one calibrated spoon of the corresponding grinded dried plant was used.

Starting from the herbal compositions listed in List 6-3 corresponding water extracts (or processed samples) were prepared according to the previously described Method B using 40 mL of water containing 100 g/l of sucrose for water extraction and including the decantation step before filtration.

The biological activity of each processed sample was determined according to the previously described Method C by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213 and ATCC 14775, with the following final dilutions of processed samples: 1:20, 1:63 and 1:200.

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions including the 2 plants of Mix[2P]-1 at 1:20 dilution is given in Table 5-1.

TABLE 5-1 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[2P]-1 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antibiofilm activity and equivalent or lower antimicrobial activity. (C) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity Planktonic Biofilm growth formation inhibition inhibition (A) Mix[2P]-1  22% 36% M[20P]-563  26% 40% M[20P]-495  25% 43% M[20P]-631  27% 42% M[20P]-465  24% 50% M[20P]-408  26% 50% M[09P]-8  31% 48% M[19P]-13  31% 50% M[18P]-12  40% 50% M[20P]-581  40% 50% M[20P]-469  40% 55% M[20P]-485  43% 52% M[20P]-427  37% 66% M[20P]-582  51% 54% M[20P]-431  44% 65% M[20P]-618  54% 57% M[20P]-580  61% 59% M[20P]-421  53% 70% M[20P]-499  60% 67% M[20P]-525  86% 77% M[2OP]-29 105% 93% (B) Mix[2P]-1  22% 36% M[20P]-723  11% 40% M[20P]-786  20% 40% M[20P]-737  22% 40% M[20P]-340  11% 40% M[20P]-382  13% 41% M[20P]-444  18% 42% M[20P]-603  12% 42% M[20P]-470  19% 42% M[20P]-503  12% 46% M[19P]-67  18% 49% M[20P]-533  21% 50% M[20P]-845  16% 51% M[20P]-377  21% 51% M[20P]-510   7% 55% (C) Mix[2P]-l  22% 36% M[17P]-9  27% 29% M[18P]-37  29% 36%

20 processed samples obtained from herbal compositions containing 9, 18, 19 or 20 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[2P]-1 (Table 5-1 A). Fourteen 20 plant mixes extracts were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 5-1 B). Two processed samples obtained from herbal compositions containing 17 and 18 plants were found to have higher antimicrobial activity and equivalent or lower antibiofilm activity (Table 5-1 C).

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions including the 2 plants of Mix[2P]-2 at 1:20 dilution is given in Table 5-2.

TABLE 5-2 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[2P]-2 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity. Planktonic Biofilm growth formation inhibition inhibition (A) Mix[2P]-2 12% −3% M[17P]-5 17% 17% M[20P]-654 16% 20% M[16P]-1 14% 22% M[17P]-1 16% 22% M[20P]-779 14% 29% M[20P]-746 15% 28% M[20P]-635 17% 26% M[20P]-844 15% 29% M[20P]-831 17% 31% M[20P]-894 18% 30% M[20P]-888 23% 25% M[20P]-407 19% 31% M[20P]-907 21% 31% M[20P]-335 19% 33% M[20P]-714 17% 35% M[20P]-668 15% 38% M[20P]-584 14% 39% M[20P]-777 16% 37% M[20P]-853 20% 34% M[20P]-785 16% 39% M[20P]-622 21% 34% M[09P]-2 17% 39% M[19P]-46 18% 37% M[20P]-387 13% 43% M[20P]-767 18% 39% M[18P]-23 24% 33% M[20P]-745 20% 37% M[20P]-478 22% 36% M[19P]-45 16% 42% M[19P]-49 19% 40% M[20P]-467 17% 42% M[20P]-396 21% 38% M[20P]-392 17% 42% M[19P]-44 20% 42% M[19P]-41 23% 40% M[19P]-48 18% 46% M[19P]-47 17% 46% M[20P]-490 20% 44% M[19P]-21 29% 35% M[20P]-632 24% 40% M[19P]-54 19% 45% M[20P]-461 19% 46% M[19P]-42 24% 41% M[20P]-506 17% 49% M[19P]-55 21% 46% M[20P]-459 26% 42% M[20P]-375 19% 52% M[19P]-2 26% 49% M[19P]-40 28% 48% M[20P]-523 28% 51% M[20P]-420 34% 56% M[20P]-429 36% 59% M[20P]-535 45% 55% M[20P]-532 42% 60% M[20P]-432 54% 72% M[20P]-555 68% 65% M[20P]-240 67% 78% M[20P]-557 86% 78% (B) Mix[2P]-2 12% −3% M[20P]-638 11% 13% M[20P]-794  6% 16% M[20P]-909  0% 19% M[20P]-538  8% 22% M[20P]-781  6% 24% M[19P]-27  7% 26% M[20P]-681 11% 24% M[16P]-3  8% 28% M[20P]-721  2% 28% M[20P]-493  7% 28% M[17P]-6  9% 29% M[20P]-659 10% 30% M[20P]-409 10% 30% M[20P]-602  4% 31% M[20P]-456  9% 31% M[20P]-383  5% 32% M[20P]-847 13% 33% M[20P]-496  6% 34% M[19P]-51 10% 34% M[20P]-371  7% 36% M[20P]-609 12% 36% M[19P]-52  9% 36% M[19P]-50 12% 36% M[18P]-24 11% 36% M[19P]-53 10% 40%

58 processed samples obtained from herbal compositions containing 9, 16, 17, 18, 19 or 20 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[2P]-2 (Table 5-2 A). 25 processed samples obtained from herbal compositions containing 16, 17, 19 or 20 plants were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 5-2 B).

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions including the 2 plants of Mix[2P]-3 at 1:20 dilution is given in Table 5-3.

TABLE 5-3 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[2P]-3 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity. Planktonic Biofilm growth formation inhibition inhibition (A) Mix[2P]-3 12% 28% M[20P]-649 16% 29% M[20P]-857 15% 32% M[20P]-703 13% 36% M[20P]-486 15% 35% M[20P]-766 13% 37% M[20P]-789 14% 36% M[20P]-725 14% 37% M[20P]-536 20% 32% M[20P]-753 14% 38% M[20P]-401 14% 38% M[20P]-765 14% 38% M[20P]-848 14% 39% M[20P]-782 14% 40% M[20P]-589 15% 39% M[20P]-607 14% 40% M[20P]-541 20% 34% M[20P]-447 15% 39% M[20P]-601 17% 38% M[20P]-494 13% 43% M[20P]-787 15% 40% M[20P]-748 15% 41% M[09P]-6 15% 41% M[20P]-480 18% 38% M[20P]-633 15% 41% M[20P]-849 14% 42% M[20P]-793 16% 42% M[18P]-9 17% 41% M[20P]-852 13% 45% M[20P]-819 20% 39% M[20P]-655 15% 44% M[20P]-455 15% 44% M[20P]-846 16% 44% M[20P]-481 18% 41% M[20P]-484 17% 42% M[20P]-590 26% 34% M[20P]-890 19% 42% M[20P]-670 21% 40% M[20P]-705 23% 39% M[20P]-816 22% 39% M[19P]-91 15% 48% M[20P]-457 22% 43% M[20P]-551 24% 41% M[19P]-100 16% 50% M[20P]-583 23% 43% M[20P]-823 22% 44% M[19P]-100 16% 50% M[19P]-98 17% 50% M[19P]-101 14% 54% M[19P]-93 14% 53% M[19P]-102 13% 54% M[19P]-5 19% 49% M[19P]-89 17% 51% M[19P]-103 13% 55% M[19P]-96 18% 52% M[20P]-475 25% 45% M[20P]-548 30% 42% M[19P]-96 19% 53% M[20P]-605 22% 50% M[20P]-577 29% 44% M[20P]-482 27% 46% M[19P]-95 19% 55% M[20P]-904 21% 55% M[20P]-559 32% 47% M[20P]-430 29% 71% M[20P]-520 62% 67% M[20P]-579 75% 66% M[20P]-560 72% 70% M[20P]-565 73% 73% M[20P]-524 81% 79% M[20P]-575 85% 76% (B) Mix[2P]-3 12% 28% M[20P]-599 11% 32% M[20P]-621 10% 32% M[20P]-740 12% 33% M[20P]-830  7% 33% M[20P]-623 12% 34% M[20P]-708  8% 35% M[20P]-880 12% 35% M[20P]-716  8% 35% M[20P]-803  4% 36% M[20P]-686 13% 36% M[20P]-663  5% 36% M[20P]-873  7% 36% M[20P]-608 10% 37% M[20P]-752 10% 37% M[20P]-443  8% 37% M[19P]-30  3% 37% M[20P]-712 12% 37% M[20P]-616  7% 38% M[20P]-724  4% 38% M[20P]-795 11% 38% M[20P]-403 13% 38% M[20P]-595  7% 38% M[20P]-771  9% 39% M[20P]-718  5% 39% M[20P]-620 12% 39% M[20P]-841  6% 40% M[20P]-804  8% 40% M[20P]-386  6% 41% M[20P]-764 11% 42% M[18P]-33 11% 42% M[20P]-349  9% 43% M[19P]-92 10% 46% M[18P]-34 11% 47% M[19P]-93 12% 50% M[18P]-31 12% 51% M[18P]-34 12% 51% M[19P]-62 10% 56%

70 processed samples obtained from herbal compositions containing 9, 19 or 20 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[2P]-3 (Table 5-3 A). 37 processed samples containing 18 to 20 plants were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 5-3 B).

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from herbal compositions including the 3 plants of Mix[3P]-1 at 1:20 dilution is given in Table 5-4.

TABLE 5-4 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on Mix[3P]-1 at 1:20 dilution. (A) Mixes extracts showing higher antimicrobial and antibiofilm activity. (B) Mixes extracts showing antimicrobial activity and equivalent or lower antibiofilm activity. Planktonic Biofilm growth formation inhibition inhibition (A) Mix[3P]-1  31% 15% M[18P]-2  35% 42% M[16P]-5  36% 41% M[14P]-2  34% 44% M[16P]-6  35% 44% M[09P]-9  33% 47% M[09P]-1  34% 45% M[18P]-7  35% 45% M[17P]-3  36% 44% M[09P]-7  35% 49% M[20P]-594  40% 46% M[09P]-6  37% 51% M[20P]-658  45% 43% M[19P]-3  34% 54% M[18P]-11  40% 49% M[19P]-104  43% 46% M[09P]-3  46% 44% M[09P]-4  46% 45% M[19P]-19  34% 57% M[18P]-13  42% 50% M[18P]-10  43% 49% M[18P]-9  42% 51% M[18P]-17  45% 51% M[20P]-634  43% 56% M[20P]-596  44% 55% M[18P]-15  47% 52% M[20P]-449  47% 56% M[09P]-10  54% 50% M[20P]-588  48% 56% M[20P]-458  50% 56% M[19P]-105  49% 56% M[13P]-2  51% 56% M[19P]-11  43% 67% M[18P]-16  54% 59% M[20P]-569  55% 60% M[20P]-296  52% 62% M[18P]-18  56% 61% M[20P]-516  61% 68% M[20P]-592  70% 71% M[20P]-626  71% 71% M[19P]-23  72% 72% M[20P]-497  72% 77% M[20P]-572  74% 78% M[20P]-574  76% 79% M[20P]-527  76% 80% M[20P]-297  83% 81% M[20P]-422  81% 83% M[20P]-564  86% 82% M[20P]-418  98% 87% M[20P]-416  99% 89% M[20P]-576 104% 86% M[20P]-573 104% 93% (B) Mix[3P]-1  31% 15% M[20P]-692  11% 22% M[20P]-612   6% 25% M[18P]-22   4% 27% M[20P]-388   9% 27% M[20P]-380  12% 28% M[11P]-12  22% 29% M[19P]-33   8% 30% M[19P]-35   6% 30% M[17P]-2  12% 30% M[20P]-678  10% 31% M[19P]-22  17% 31% M[20P]-552  16% 32% M[17P]-4   9% 32% M[20P]-806  12% 32% M[12P]-1  19% 33% M[14P]-1  18% 33% M[19P]-28   6% 33% M[12P]-2  22% 34% M[18P]-1  16% 34% M[19P]-36   6% 34% M[20P]-479  23% 34% M[15P]-1  23% 34% M[09P]-21  22% 34% M[20P]-446  10% 34% M[19P]-34   7% 34% M[19P]-106  29% 35% M[10P]-41   8% 35% M[13P]-1  22% 35% M[18P]-5  18% 35% M[19P]-39  -1% 35% M[11P]-3  12% 35% M[20P]-439  19% 36% M[11P]-2  13% 36% M[18P]-20   6% 36% M[20P]-757  18% 36% M[10P]-303  25% 36% M[11P]-11  29% 37% M[11P]-6   9% 37% M[19P]-37   5% 37% M[18P]-19  10% 37% M[18P]-4  29% 37% M[18P]-35  27% 37% M[18P]-25  11% 37% M[11P]-5  10% 38% M[20P]-694  20% 38% M[11P]-8  10% 38% M[11P]-9  11% 38% M[20P]-733  12% 38% M[11P]-7  10% 38% M[19P]-31   6% 38% M[19P]-32   4% 38% M[19P]-29   6% 38% M[18P]-6  22% 38% M[20P]-374   2% 38% M[11P]-4  12% 39% M[20P]-791  13% 39% M[20P]-758  15% 39% M[19P]-26   6% 39% M[20P]-697  17% 39% M[19P]-38   4% 40% M[19P]-24  28% 40% M[16P]-2  24% 40% M[18P]-21   9% 40% M[15P]-5  29% 40% M[17P]-8  30% 40% M[20P]-613   8% 41% M[18P]-14  27% 41% M[20P]-604  14% 41% M[11P]-1  17% 41% M[18P]-36  29% 41% M[18P]-3  23% 42% M[20P]-453   5% 42% M[19P]-25   9% 42% M[20P]-709  18% 42% M[20P]-474  16% 45% M[20P]-666  22% 45% M[20P]-902  21% 46% M[20P]-435  15% 46% M[20P]-567  30% 47% M[19P]-4  24% 47% M[19P]-16  18% 47% M[19P]-59  10% 48% M[19P]-17  23% 48% M[19P]-58  20% 49% M[19P]-60  13% 49% M[19P]-15  27% 50% M[11P]-10  31% 50% M[19P]-14  27% 50% M[19P]-6  32% 51% M[20P]-445  21% 51% M[19P]-8  30% 51% M[19P]-18  21% 52% M[19P]-9  29% 52% M[19P]-7  30% 52% M[19P]-10  29% 52% M[19P]-68  23% 53% M[19P]-63  16% 53% M[19P]-69  22% 53% M[19P]-1  26% 54% M[19P]-66  24% 54% M[19P]-12  26% 54% M[20P]-472  27% 54% M[19P]-20  31% 54% M[19P]-70  19% 55% M[19P]-62  17% 56% M[19P]-57  21% 56% M[19P]-65  27% 57% M[19P]-73  18% 57% M[20P]-514  30% 58% M[19P]-56  24% 58% M[19P]-64  27% 59% M[19P]-72  20% 60% M[19P]-71  19% 61%

51 processed samples obtained from herbal compositions containing 9, 16, 17, 18, 19 or 20 plants were found to have higher antibiofilm and antimicrobial activity than the reference mix extract Mix[3P]-1 (Table 5-4 A). 113 processed samples containing 9 to 20 plants were found to have higher antibiofilm activity and equivalent or lower antimicrobial activity (Table 5-4 B).

Comparative Example 3

As a comparative Example, we report several 20 plant mixes extracts containing only one plant among the 3 plants of Example 1 (PA13, PA21, PB12), which have been found to have none or poor antimicrobial and antibiofilm activities against Staphylococcus aureus.

Ten herbal compositions having the content reported in List 5-8 were prepared according to the previously described Method A.

List 5-8: Herbal Compositions of Processed Samples

M[20P]-39: PB00; PB01; PB02; PB03; PB04; PB05; PB06; PB07; PB08; PB09; PB10; PB11; PB12; PB13; PB14; PB15; PB16; PB17; PB18; PB19;

M[20P]-239: PA00; PA05; PA21; PA23; PA26; PA33; PA39; PA60; PA85; PB00; PB06; PB32; PB79; PC02; PC26; PC34; PC38; PC62; PC78; PC84;

M[20P]-337: PA00; PA10; PA11; PA15; PA18; PA21; PA22; PA24; PA26; PA28; PA52; PA94; PA98; PB10; PB20; PB26; PB29; PB82; PC67; PC97;

M[20P]-362: PA21; PA23; PA25; PA33; PA37; PA57; PA69; PA81; PA92; PB16; PB25; PB26; PB28; PB29; PB78; PC20; PC21; PC71; PC83; PD02;

M[20P]-364: PA15; PA21; PA25; PA28; PA39; PA75; PA80; PA85; PA89; PA98; PB01; PB06; PB24; PB28; PB35; PC24; PC25; PC57; PC75; PC82;

M[20P]-540: PA06; PA10; PA12; PA14; PA20; PA21; PA26; PA27; PA27; PA57; PA57; PA57; PA69; PB07; PB82; PC11; PC20; PC23; PC55; PC98;

M[20P]-647: PA10; PA11; PA24; PA26; PA28; PA33; PA35; PA40; PA57; PA69; PB00; PB07; PB12; PB28; PB33; PB38; PB60; PC55; PC67; PC97;

M[20P]-652: PA00; PA07; PA13; PA15; PA16; PA23; PA26; PA28; PA33; PA39; PA57; PA92; PB28; PB38; PC20; PC21; PC52; PC55; PC74; PC97;

M[20P]-862: PA11; PA15; PA18; PA21; PA25; PA27; PA28; PA29; PA35; PA38; PA52; PA86; PA89; PA92; PB07; PB28; PB88; PC11; PC55; PC98;

M[20P]-872: PA13; PA14; PA16; PA28; PA29; PA30; PA33; PA35; PA39; PA81; PA90; PA92; PB00; PB32; PC12; PC34; PC37; PC67; PC97; PD02.

Ten water extracts (or processed samples) were prepared from the corresponding herbal compositions according to the previously described Method B using 40 mL of water containing 100 g/l of sucrose for water extraction and including the decantation step before filtration.

For each processed sample the biological activity was determined according to the previously described Method C, with the following final dilutions of processed samples: 1:20, 1:63 and 1:200.

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples at 1:20 dilution is given in Table 5-5.

TABLE 5-5 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed samples at 1:20 dilution. Planktonic growth Biofilm formation inhibition inhibition Mix[2P]-1 22% 36% Mix[2P]-2 12% −3% Mix[2P]-3 12% 28% M[20P]-362 −1% −6% M[20P]-364 −5%  6% M[20P]-039  1%  6% M[20P]-862  5%  2% M[20P]-337  0%  9% M[20P]-872  2% 11% M[20P]-540  5% 10% M[20P]-652  9%  7% M[20P]-239  9%  7% M[20P]-647  9%  9%

All tested processed samples obtained from herbal compositions containing 20 plants including only one plant among the 3 plants of Example 1 (PA13, PA21, PB12) showed lower antimicrobial activity than the three 2 plant mixes extracts of reference Mix[2P]-1 (obtained from PA13 and PA21), Mix[2P]-2 (obtained from PA21 and PB12) and Mix[2P]-3 (obtained from PA13 and PB12) and lower antibiofilm activity than Mix[2P]-1 and Mix[2P]-3.

Example 6

In this Example, we report the simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus pseudintermedius of 2 processed samples chosen among the best mixes described in Example 4 and 5.

The dried plants of pharmaceutical grade were obtained from “Pharmacie Fontgiève” (Clermont-Ferrand, France) and “Pharmacie St Herem” (Clermont-Ferrand, France).

Two herbal preparations containing the dried plant powders reported in the Table 6-1 below, were prepared according to the previously described Method A.

TABLE 6-1 Herbal compositions M[20P]-576 M[10P]-154 Plant 1 2 spoons of PA11 1 spoon of PA11 Plant 2 1 spoon of PA12 1 spoon of PA12 Plant 3 3 spoons of PA13 1 spoon of PA13 Plant 4 2 spoons of PA14 1 spoon of PA21 Plant 5 1 spoon of PA20 1 spoon of PA22 Plant 6 1 spoon of PA21 1 spoon of PA79 Plant 7 1 spoon of PA22 1 spoon of PB12 Plant 8 1 spoon of PA29 1 spoon of PB49 Plant 9 1 spoon of PA39 1 spoon of PC29 Plant 10 1 spoon of PB12 1 spoon of PC63 Plant 11 1 spoon of PB60 Plant 12 1 spoon of PC20 Plant 13 1 spoon of PC26 Plant 14 1 spoon of PC33 Plant 15 1 spoon of PC37 Plant 16 1 spoon of PC49

Starting from such herbal compositions 2 corresponding water extracts (or processed samples) were prepared according to the previously described Method B using 40 mL for M[20P]-576 and 20 mL for M[10P]-154 of water containing 100 g/l of sucrose for water extraction and including the centrifugation step before filtration.

The biological activity of the processed samples was determined according to the previously described Method C by using the following strains:

    • Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213, ATCC 14775, NCTC 12493, ATCC 33591, ATCC 33592, ATCC 43300, ATCC 700698, ATCC 700699, ATCC 9144 and ATCC BAA-44
    • Staphylococcus epidermidis strains: ATCC 14990, ATCC 12228, ATCC 700296, ATCC 49134 and ATCC 49461
    • Staphylococcus pseudintermedius strain: ATCC 49444

The minimum inhibitory concentration (MIC) observed with the 2 processed samples is given in Table 6-2 in the form of HID, as well as the average efficacy over all the strains of Staphylococcus aureus and Staphylococcus epidermidis.

TABLE 6-2 HID of the 2 processed samples on 14 strains of Staphylococcus aureus, 5 strains of Staphylococcus epidermidis and 1 strain of Staphylococcus pseudintermedius M[10P]-154 M[20P]-576 S. aureus ATCC 25923 20 20 ATCC 49476 40 40 ATCC 6538 10 10 ATCC 51740 20 40 ATCC 29213 20 20 ATCC 14775 20 20 NCTC 12493 40 40 ATCC 33591 20 40 ATCC 33592 20 40 ATCC 43300 20 40 ATCC 700698 20 20 ATCC 700699 40 20 ATCC 9144 20 20 ATCC BAA-44 40 20 Average 25 28 S. epidermidis ATCC 14990 40 20 ATCC 12228 40 20 ATCC 700296 40 20 ATCC 49461 40 20 ATCC 49134 80 80 Average 48 32 S. pseudintermedius ATCC 49444 40 40 Total average 32 30

M[10P]-154 and M[20P]-576 showed full antimicrobial activity on all tested Staphylococcus aureus and Staphylococcus epidermidis. M[10P]-154 showed full antimicrobial activity at 1:80 on 1 Staphylococcus epidermidis strain, at 1:40 on 4 Staphylococcus aureus strains, 4 Staphylococcus epidermidis and the tested strain of Staphylococcus pseudintermedius, at 1:20 on 9 Staphylococcus aureus strains, and at 1:10 on 1 Staphylococcus aureus strain.

M[20P]-576 showed full antimicrobial activity at 1:80 on 1 Staphylococcus epidermidis strain, at 1:40 on 6 Staphylococcus aureus strains and the tested strain of Staphylococcus pseudintermedius, at 1:20 on 7 Staphylococcus aureus strains and 4 Staphylococcus epidermidis and at 1:10 on 1 Staphylococcus aureus strain.

The bacteria planktonic growth inhibition percentage and the biofilm formation inhibition (IBF) percentage of the 2 processed samples at 1:20 dilution is given in Table 6-3, as well as the average efficacy over all the strains of Staphylococcus aureus and Staphylococcus epidermidis.

TABLE 6-3 planktonic growth inhibition and biofilm formation inhibition (IBF) of the 2 processed samples on 14 strains of Staphylococcus aureus, 5 strains of Staphylococcus epidermidis and 1 strain of Staphylococcus pseudintermedius at 1:20 dilution planktonic biofilm growth formation inhibition inhibition (IBF) M[10P]- M[20P]- M[10P]- M[20P]- 154 576 154 576 S. aureus ATCC 25923 96%  99% 90% 88% ATCC 49476 93% 109% 93% 95% ATCC 6538 49%  56% 45% 60% ATCC 51740 91% 106% 89% 96% ATCC 29213 94% 105% 91% 92% ATCC 14775 90% 105% 88% 90% NCTC 12493 99% 113% 93% 92% ATCC 33591 95% 113% 91% 93% ATCC 33592 97% 107% 91% 93% ATCC 43300 94% 110% 90% 92% ATCC 700698 92% 113% 89% 91% ATCC 700699 88%  95% 98% 96% ATCC 9144 82%  91% 88% 84% ATCC BAA-44 90% 101% 93% 94% Average 89% 102% 88% 90% S. epidermidis ATCC 14990 90% 101% 91% 90% ATCC 12228 90% 101% 93% 91% ATCC 700296 86%  90% 81% 82% ATCC 49461 90%  99% 93% 85% ATCC 49134 89%  96% 96% 90% Average 89%  98% 91% 88% S. pseud- ATCC 49444 92% 103% 95% 93% intermedius Total average 89% 101% 89% 89%

At 1:20 dilution, M[10P]-154 showed an average of 88% of biofilim formation inhibition on all tested Staphylococcus aureus strains whereas M[20P]-576 showed an average of 90%. The two mixes showed lower antimicrobial activity on one strain: ATCC 6538 (45% for M[10P]-154, 60% for M[20P]-576). M[10P]-154 showed an average of 91% of planktonic growth inhibition on all tested Staphylococcus epidermidis strains whereas M[20P]-576 showed an average of 88%. (cf Table 6-3). The two mixes showed more respectively 95% (M[10P]-154) and 93% (M[20P]-576) of inhibition percentage on the Staphylococcus pseudintermedius tested. Thus, M[10P]-154 and M[20P]-576 have demonstrated antibiofilm activity on Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus pseudin term edius at 1:20 dilution.

Example 7

Solution of Mix[20C]-576 (described in Example 6) used for the following formulations was prepared according to the previously described Method A and B with the exception of the filtering step, which is in this Example performed by suction filtration with a Büchner funnel filled with 5 cm thick hydrophilic cotton layer. For clarity, this cotton filtered solution will be referred as SK576 in the following Example.

An ointment and two creams to be used for topical applications were prepared with an emulsification method as follows:

    • Phase A and Phase B were prepared by mixing with orbital shaking in two separate beakers the following ingredients:

Phase A Phase B Ointment - 73.8 g of organic almond - 29.6 g of SK576, oil (Hyteck, prepared as disclosed in “Aroma Zone ™”, France) Example 6 - 18.5 g of organic - 13.5 g of organic beeswax (Hyteck, glycerin (The Naissance “Aroma Zone ™”, France) Trading & Innovation, - 9.7 g of organic shea “Naissance ™”, UK) butter (Hyteck, - 2.7 g of Ascorbic “Aroma Zone ™”, France) acid (Hyteck “Aroma -1.0 mL of natural Zone ™”, France) tocopherol (Hyteck, “Aroma Zone ™”, France) Cream 1 - 30.4 g of organic - 51.5 g of SK576, almond oil (Hyteck, prepared as disclosed in “Aroma Zone”, France): Example 6 - 8.3 g of organic - 14.9 g of organic Olivem ® 1000 glycerin (The Naissance (Hyteck, “Aroma Trading & Innovation Zone ™”, France) “Naissance ™”, UK) - 3.1 g of menthol (Hyteck “Aroma Zone ™”, France) - 1.0 mL of natural tocopherol (Hyteck, “Aroma Zone ™”, France) Cream 2 - 14.9 g of organic - 160.0 g of SK576, almond oil (Hyteck prepared as disclosed in “Aroma Zone ™”, France): Example 6 - 11.8 g of organic - 10 g of organic glycerin Olivem ® 1000 (The Naissance (Hyteck “Aroma Trading & Innovation, Zone ™”, France) “Naissance ™”, UK) - 5.1 g of organic shea butter (Hyteck “Aroma Zone ™”, France) - 1.0 mL of natural tocopherol (Hyteck “Aroma Zone ™”, France)
    • The two beakers were heated to 70° C. using a warm water bath and maintained at 70° C. during 10 min to avoid the crystallization of beeswax.
    • Phase B was then slowly poured into Phase A, keeping the beaker in the warm water bath and with constant stirring by a mechanical immersion blender (Moulinex “Infinyforce_hand blender”, France).
    • The beaker containing the mix of phase A and B was then removed from the warm bath and the ointment was formed by using an immersion “mayonnaise blender” (Moulinex “Infinyforce_hand blender”, France).

A gel to be used for topical applications was prepared by

    • Filtrating 20.6 g of SK576, on an hydrophilic syringe filter (0.22 μm).
    • Adding 0.75 g of Xanthan powder (Hyteck “Aroma Zone™”, France) and 0.25 g of ascorbic acid (Hyteck “Aroma Zone™”, France) to the filtered solution and mix with a spatula for 5-10 min such as to form.

A spray solution to be used for topical applications was prepared by:

    • Mixing 5.5 g of SK576 with 11.9 g of absolute ethanol (Sigma-Aldrich, France) in a 20 mL beaker which were stored in a 5° C. refrigerator for 3H.
    • Placing the beaker with the cold mixture in an ice-water bath and gently adding 0.7 g of Kolliphor® P-407 (Sigma-Aldrich, France) under agitation with a magnetic bar and stirrer.
    • Following 30 min agitation under ice-water bathing, the solution was filled into a reagent sprayer (CAMAG, Switzerland) for administration.

The protocol of each of these five preparations can be easily scaled to different volumes of preparation by keeping the same relative amount of each ingredient.

Example 8

Biological Activity of a Water Extract of the Invention in a Mouse Model of Lethal Staphylococcus aureus Infection

INTRODUCTION

S. aureus strain Newman was isolated in 1952 from a human infection and has been used extensively in animal models of staphylococcal infections due to its robust virulence phenotype.

Female OF1 mice have been extensively used to study the pathogenesis induced by Staphylococcus aureus. Early work on the pathogenesis of Staphylococcus aureus infections used the intraperitoneal route of challenge to infect and treat mice because of its technical ease.

The aim of this Example was to evaluate the antimicrobial activity of a water extract of the invention in female OF1 mice infected with lethal dose of Newman Staphylococcus aureus. The treatment was administered by oral route according to the schedule Q1Dx10, 2 days before infection to 7 days after infection. Monitoring of mortality was performed from D0 to D15.

Vehicle was used as negative control.

Material and Methods

Test Substance and Controls

Test Substance: water extract M[20C] 576 (obtained starting from 120 g/l of herbal composition, prepared as described in Example 6). It was provided in 11 vials containing 1,200 μl each of water extracts. It was stored at −20° C.

Vehicle: sucrose 10% water solution. It will be stored at −20° C.

Bacterial strain: Newman Staphylococcus strain (provided by Dr T. Foster, Trinity College, Dublin, Ireland, Batch No. 14156, LST4).

Animal Purchasing and Caging

Twenty (20) Crl:OF1 (outbred) female mice, 6 weeks old were purchased from Charles River (L'Arbresle, France).

The animal care unit was authorized by the French Ministries of Agriculture and Research (Agreement Number B35-288-1). Animal experiments were performed according to ethical guidelines of animal experimentations (Principes d'éthique de l'expérimentation animale, Directive n° 86/609 CEE du 24 Nov. 1986, Décrêt n° 87/848 du 19 Oct. 1987, Arrêté d'Application du 19 Avril 1988).

The animals were maintained in “A2” rooms under controlled conditions of temperature (22±3° C.), humidity (50±20%), photoperiod (12 h light/12 h dark), air exchange and low pressure. The air handling system is programmed for 14 air changes an hour, with no recirculation.

Food and water were provided ad libitum, being placed in the metal lid on top of the cage.

Treatment and Exnerimental Design

Preparation of Bacteria—Amplification of Bacteria for Challenge

One vial of bacteria was defrosted and 100 μl of inoculum was cultured in 50 ml of Tryptic Soy Broth (TSB, Ref. 43592 or 22902, Sigma). Bacteria were incubated at 37° C. during 20 h under 110 rpm agitation on horizontal shaker. Bacterial suspension will be diluted at 1/20 (100 μl+1.9 ml of TSB) and optical density will be read at 680 nm (O.D.680 nm).

Then, 10 ml of bacteria was centrifuged at 5,200 rpm (3400 g) during 10 min at 4° C. Supernatant was discarded and bacteria was solubilized in 10 ml of DPBS (Ref. BE17-512F, Lonza, France). Bacterial suspension (CO) was diluted at 1/20 (100 μl+1.9 ml of DPBS) and O.D.680 nm will be measured.

The O.D.680 nm measured before and after centrifugation must be similar.

The concentration of CFU/ml of the bacteria solution was calculated as follow:


CFU/ml=(O.D.680 nm×20(dilution factor)×6.5·108 (CFU/U.D.O)

Mucin Preparation

Mucin (Sigma, France) was prepared at 20% 14-16 days before treatment as described below:

DPBS (Dulbecco's Phosphate-Buffered Saline) was pre-warmed at 56° C. and ¾ of the final volume was transferred in a mixer, then hog mucin will be added and mixed. The mixer was washed with the remaining volume of DPBS. Then, the 20% mucin solution was transferred in an Erlenmeyer Flask and was incubated at 37° C. under agitation during 1 hour and 30 minutes. The ¾ of 20% mucin solution was transferred in a sterile bottle. The remainder ¼ of solution was stocked separately. Then the ¾ and the ¼ solutions was sterilized by autoclaving. Sterile 20% hog mucin solution was stored at +4° C.

The day before the infection, the pH of ¾ of 20% mucin solution was adjusted at 7.4 by adding filtered NaOH 30%. If necessary, a part of the ¼ remaining solution was used to adjust pH.

Bacteria Preparation

According to O.D.680 nm results, bacteria (CO) were diluted in DPBS to reach the concentration of 6.8×107 CFU/ml. These suspensions were then diluted at % in 20% hog mucin to get the LD80 concentration of 3.4×107 CFU/ml in PBS-10% hog mucin. The suspension used for treatment was put in tube containing sterile glass beads (4 mm, Ref. 068502, Dutscher, France). Suspension were not vortexed, but mixed by inverting the tubes. Bacteria were placed on ice before treatments and kept on ice during treatment.

CFU Determination

To determine the exact concentration of the bacterial suspension before and after inoculated into the animals, the bacterial suspension was diluted in 10-fold serial dilutions in DPBS-0.5% Tween 20 (Sigma, France) as described in Table 8-1 in deep wells (50 μl of bacteria+450 μl of DPBS-0.5% Tween 20). Then, 6×50 μl of bacterial dilutions 3, 4, 5 and 6 were seeded on Tryptic Soy Agar plate (Tryptic Soy Agar, Sigma) and placed at 37° C. overnight. Colonies were counted to determine the real CFU (Colony-Forming Units) of inoculums.

The numeration was realized on the drop (50 μl) that contain between 20 and 100 colonies. The shaded boxes correspond to the dilutions seeded on plates.

TABLE 8-1 Dilutions of the bacterial suspension in order to determine the real CFU (viability) of inoculums. The shaded boxes correspond to the dilutions seeded on plates. Bacterial overnight Dilutions suspension 1 2 3 4 5 6 in PBS 1/10 1/100 1/103 1/104 1/105 1/106 For example, 3.4 × 106 3.4 × 105 34000 3400 340 34 initial concentration: 3.4 × 107 CFU/ml Number of 1.7 × 105 1.7 × 104 1.7 × 103 170 17 1.7 colonies/50 μl (1 drop)

Randomization

Before experiment (D-2), 20 healthy OFI female mice were randomized in 2 groups of 10 mice/group according to body weight criteria, so that the mean body weights of groups was not statistically different.

Treatments

Treatments were performed from D-2 to D7 (D0: day of bacteria inoculation) by oral (PO) administration. Bacterial inoculations were done by intraperitoneal injection (IP).

Inoculation

A D0, mice were inoculated with bacterial suspension. Each inoculation consisted in an intraperitoneal injection of 500 μl of the suspension at 3.4×107 CFU/ml (1.7×107 CFU/mouse).

Experimental Design

Animals were treated with an oral administration of the Test Substance from D-2 to D7. At D0, treatments were performed 1 h after bacteria inoculation.

The experimental groups will be defined as described below and summarized in Table 8-2.

    • The 10 mice of the group 1 will be treated from D-2 to D7 with the Vehicle by oral administration according to the treatment schedule Q1DX10.
    • The 10 mice of the group 2 will be treated from D-2 to D7 with the Test Substance by oral administration according to the treatment schedule Q1DX10.

TABLE 8-2 Experimental design Number Test Administration Administration Treatment Groups of mice substance route volume schedule Bacterial model 1 10 Vehicule PO 10 mL/kg Q1D × 10 IP lethal Newman 2 Test (from D-2 to D7) Staphylococcus Substance aureus (3.4 × 107 CFU/ml) 500 μl

Monitoring of Animals

From D0 to D1, mice were observed twice a day for mortality.

From D2 to D15, mice were observed once a day for mortality.

The body weight of mice was recorded twice a week until the end of the experiment.

Detailed clinical observations (changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, respiratory function, changes in gait, posture) was monitored twice a week as described in Table 8-3 below for example. Description of this list is not exhaustive and was complemented by other observations, if necessary.

TABLE 8-3 Clinical observations/parameters Para- De- Para- De- Para- De- meters scription meters scription meters scription Mobility/ Stationary Behavior Fearful Fur Normal Gait Reduced Normal Spiked Normal Aggressive Loss Excessive Breathing Slow Colored Carriage Normal Normal Not Prostrate Speed applicable Paralysis No Irregular Mucous Normal Front right Eyes Normal membranes Abnormal Front left Tearful (nose) Hind right Closed Abnormal Hind left Skin Normal (mouth) Right Redness Abnormal Left Patch (nose/ Front Wound mouth) Hind Hematoma Excretions Few Normal Abundant Dry

Sacrifice of Mice

At D15, survived mice were sacrificed by exsanguinations, followed by cervical dislocation, if needed. No autopsy was performed.

Results

The analysis of mice survival showed the results reported in the following Tables 8-4 and 8-5.

TABLE 8-4 Number of mice survived per day Number of survived mice per day N N N N N N N N N Groups D0 D1 D2 D3 D4 D5 D6 D7 D8 Group 1 10 6 6 5 5 3 2 NA 0 (Vehicle, PO, Q1D × 10, 10 ml/kg) Group 2 10 6 6 6 6 6 4 NA 3 (TS pure, PO, Q1D × 10, 10 ml/kg)

TABLE 8-5 Percentage of survived mice per day Percentage of survival N N N N N N N N N Groups D0 D1 D2 D3 D4 D5 D6 D7 D8 Group 1 100% 60% 60% 50% 50% 30% 20% NA  0% (Vehicle, PO, Q1D × 10, 10 ml/kg) Group 2 100% 60% 60% 60% 60% 60% 40% NA 30% (TS pure, PO, Q1D × 10, 10 ml/kg) NA = Not Available

The treatment allowed to reduce the mortality of mice from 100% to 70%. This animal study thus showed that the claimed antimicrobial and anti-biofilm activities observed in-vitro is translated into a therapeutic effect, when the water extract of the invention is administered via oral route to animals.

Example 9

In this Example we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus of the disclosed composition of 2 plants among 3: Filipendula ulmaria PA13, Camellia sinensis PA21, Arctostaphylos uva-ursi PB12 and at least 1 plant among 3: Eugenia caryophyllus PC20, Vitis vinifera var. tinctorial PA22, Desmodium adscendens PB07. The dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France).

Starting from such three different plant powders six herbal compositions were prepared according to the Method A reported before.

List 9-1: Herbal Compositions of Processed Samples

M[02P]-064: PA13; PA21;

M[03P]-050: PA13; PA21; PC20;

M[03P]-053: PA13; PA21; PB07;

M[03P]-056: PA13; PA21; PA22;

M[02P]-065: PA13; PB12;

M[03P]-051: PA13; PB12; PC20;

M[03P]-054: PA13; PB12; PB07;

M[03P]-057: PA13; PB12; PA22;

M[02P]-066: PA21; PB12;

M[03P]-052: PA21; PB12; PC20;

M[03P]-055: PA21; PB12; PB07;

M[03P]-058: PA21; PB12; PA22;

M[03P]-059: PA13; PB12; PA21;

M[04P]-025: PA13; PB12; PA21; PC20;

M[04P]-026: PA13; PB12; PA21; PB07;

M[04P]-027: PA13; PB12; PA21; PA22.

Starting from the herbal compositions listed in List 6-3 corresponding water extracts (or processed samples) were prepared according to the previously described Method B using 40 mL of water containing 100 g/l of sucrose for water extraction and including the decantation step before filtration.

The biological activity of each processed sample was determined according to the previously described Method C by using the following Staphylococcus aureus strains: ATCC 6538, with the following final dilutions of processed samples: 1:5 for the biofilm formation inhibition and 1:10 for the growth inhibition. Note that growth inhibition is close to 100% for each mix at 1:5 dilution, and biofilm formation inhibition is close to 0% for all mixes at 1:10 dilution.

The bacteria planktonic growth inhibition percentage and biofilm formation inhibition percentage over all the tested strain of the processed samples obtained from the different herbal compositions are reported in the following Table 9-1.

TABLE 9-1 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the Staphylococcus aureus strain ATCC ® 6538 ™ of the processed sample based on the compositions given in List 9-1. The processed samples were used at a 1:10 dilution for the planktonic growth inhibition tests and at a dilution of 1:5 for the biofilm formation inhibition. Planktonic Biofilm Planktonic Biofilm growth formation growth formation (A) inhibition inhibition (B) inhibition inhibition M[02P]-064 47% 34% M[02P]-065 42% 15% M[03P]-050 70% 56% M[03P]-051 59% 36% M[03P]-053 55% 35% M[03P]-054 54% 22% M[03P]-056 56% 38% M[03P]-057 54% 34% Planktonic Biofilm Planktonic Biofilm growth formation growth formation (C) inhibition inhibition (D) inhibition inhibition M[02P]-066 40% 23% M[03P]-059 59% 87% M[03P]-052 53% 28% M[04P]-025 69% 90% M[03P]-055 59% 27% M[04P]-026 64% 90% M[03P]-058 60% 33% M[04P]-027 64% 89%

(A) lists extracts obtained from herbal compositions containing PA13 and PA21, in which the addition of one among PC20, PB07 and PA22 improves the activity; (B) lists extracts obtained from herbal compositions containing PA13 and PB12, in which the addition of one among PC20, PB07 and PA22 improves the activity; (C) lists extracts obtained from herbal compositions containing PA13, PA21 and PB12, in which the addition of one among PC20, PB07 and PA22 improves the activity; (D) lists extracts obtained from herbal compositions containing PA13 and PA21, in which the addition of one among PC20, PB07 and PA22 improves the activity.

Example 10

In this Example, we illustrate simultaneous antimicrobial and antibiofilm activities against Staphylococcus aureus of 324 mixtures extracts obtained from the disclosed herbal compositions comprising 2 plants among 3: Filipendula ulmaria (PA13), Camellia sinensis (PA21), Arctostaphylos uva-ursi (PB12) and at least 1 plant among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctorial (PA22), Desmodium adscendens (PB07).

For the mixes from M[20C]-2083 to M[20C]-20798, the dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France). For the mixes from M[20C]-2805 to M[20C]-2957, the dried plants of pharmaceutical grade were obtained from “Pharmacie Fontgiève” (Clermont-Ferrand, France, plants PA00 to PB12) and “Pharmacie St Herem” (Clermont-Ferrand, France, plants PB13 to PD02).

Herbal compositions containing from 3 to 20 plant powders were prepared according to the previously described Method A.

Starting from the herbal compositions, corresponding water extracts (or processed samples) were prepared according to the previously described Method B using 40 mL of water containing 100 g/l of sucrose for water extraction and including the decantation step before filtration.

The biological activity of each processed sample was determined according to the previously described Method C by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213 and ATCC 14775, with the 1:20 final dilutions of processed samples.

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from the following herbal compositions of, respectively, List 10-1, List 10-2, List 10-3 and List 10-4 is given in, respectively, Table 10-1, Table 10-2, Table 10-3 and Table 10-4. In each set, the extracts obtained from herbal compositions containing more than 3 or 4 plants were found to exhibit planktonic growth inhibition or biofilm formation inhibition (or both inhibitions) greater than any of the mixes containing only 3 or 4 plants

List 10-1: Extracts from herbal compositions containing 2 plants among 3: Filipendula ulmaria (PA13), Camellia sinensis (PA21) and at least one among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctorial (PA22), Desmodium adscendens (PB07):

M[03P]-050: PA13; PA21; PC20;

M[03P]-053: PA13; PA21; PB07;

M[03P]-056: PA13; PA21; PA22;

M[20C]-2808: PA11; PA12; PA13; PA13; PA18; PA21; PA23; PA24; PA28; PA69; PB07; PB88; PC23; PC24; PC49; PC52; PC55 (2 spoons); PC60; PC97;

M[20C]-2820: PA10; PA11 (2 spoons); PA12; PA13; PA14; PA18; PA20; PA21; PA25; PA29; PA33; PA35; PA67; PA75; PB07; PB60; PC33; PC35; PC55;

M[20C]-2821: PA11; PA13 (2 spoons); PA19; PA20; PA21; PA22; PA25; PA29; PA33; PA35; PA75; PB07; PB36; PC20; PC33 (2 spoons); PC49; PC55; PC97;

M[20C]-2826: PA11; PA13 (2 spoons); PA20; PA21; PA24; PA25; PA29; PA33; PA34; PA35; PA52; PA70; PA75; PB07; PC11; PC21; PC33; PC37; PC74;

M[20C]-2827: PA10; PA11; PA13 (2 spoons); PA20; PA21; PA25; PA26; PA29; PA33; PA35; PA57; PA75; PB02; PB07; PB28; PC11; PC23; PC33; PC49;

M[20C]-2830: PA10; PA11; PA12; PA13; PA14; PA18; PA21; PA22; PA25; PA29; PA32; PA34; PA67; PB07; PB60; PB88; PC33; PC35; PC55; PC98;

M[20C]-2831: PA13; PA13; PA19; PA21; PA22; PA22; PA25; PA29; PA32; PA34; PB07; PB36; PB88; PC20; PC33; PC33; PC49; PC55; PC97; PC98;

M[20C]-2833: PA10; PA11; PA13; PA14; PA21; PA22; PA24; PA25; PA26; PA29; PA32; PA34; PB33; PB88; PB88; PC12; PC23; PC27; PC33; PC98;

M[20C]-2836: PA13; PA13; PA21; PA22; PA24; PA25; PA29; PA32; PA34; PA34; PA52; PA70; PB07; PB88; PC11; PC21; PC33; PC37; PC74; PC98;

M[20C]-2837: PA10; PA13 (2 spoons); PA21; PA22; PA25; PA26; PA29; PA32; PA34; PA57; PB02; PB07; PB28; PB88; PC11; PC23; PC33; PC49; PC98;

M[20C]-2838: PA12; PA13 (2 spoons); PA18; PA21; PA21; PA22; PA24; PA25; PA29; PA32; PA34; PB88; PC23; PC33; PC49; PC52; PC55; PC97; PC98;

M[20C]-2839: PA13 (2 spoons); PA14; PA21; PA22; PA25; PA25; PA26; PA29; PA32; PA34; PA39; PA52; PA57; PB33; PB88; PC33; PC71; PC97; PC98;

M[20C]-2858: PA12; PA13; PA18; PA20; PA21; PA23; PA24; PA24; PA67; PB07; PB28; PB33; PC12; PC20; PC21; PC23; PC49; PC52; PC55; PC97;

M[20C]-2861: PA13; PA19; PA21; PA22 (2 spoons); PB07; PB28; PB36; PB60; PC11; PC20; PC20; PC23; PC33; PC49; PC55 (2 spoons); PC60; PC74; PC97;

M[20C]-2866: PA13; PA21; PA22; PA24; PA34; PA52; PA70; PB07; PB28; PB60; PC11 (2 spoons); PC20; PC21; PC23; PC37; PC55; PC60; PC74 (2 spoons);

M[20C]-2867: PA10; PA13; PA21; PA22; PA26; PA57; PB02; PB07; PB28; PB28; PB60; PC11 (2 spoons); PC20; PC23; PC23; PC49; PC55; PC60; PC74;

M[20C]-2868: PA12; PA13; PA18; PA21; PA21; PA22; PA24; PB28; PB60; PC11; PC20; PC23 (2 spoons); PC49; PC52; PC55 (2 spoons); PC60; PC74; PC97;

M[20C]-2869: PA13; PA14; PA21; PA22; PA25; PA26; PA39; PA52; PA57; PB28; PB33; PB60; PC11; PC20; PC23; PC55; PC60; PC71; PC74; PC97;

M[20C]-2871: PA10; PA11; PA13; PA19; PA21; PA22; PA35; PA57; PB07; PB07; PB36; PB77; PC20 (2 spoons); PC23; PC33; PC49; PC55; PC71; PC97;

M[20C]-2876: PA10; PA11; PA13; PA21; PA24; PA34; PA35; PA52; PA57; PA70; PB07 (2 spoons); PB77; PC11; PC20; PC21; PC23; PC37; PC71; PC74;

M[20C]-2877: PA10; PA10; PA11; PA13; PA21; PA26; PA35; PA57; PA57; PB02; PB07; PB28; PB77; PC11; PC20; PC23 (2 spoons); PC49; PC71;

M[20C]-2878: PA10; PA11; PA12; PA13; PA18; PA21; PA21; PA24; PA35; PA57; PB07; PB77; PC20; PC23; PC23; PC49; PC52; PC55; PC71; PC97;

M[20C]-2879: PA10; PA11; PA13; PA14; PA21; PA25; PA26; PA35; PA39; PA52; PA57 (2 spoons); PB07; PB33; PB77; PC20; PC23; PC71 (2 spoons); PC97;

M[20C]-2889: PA10; PA12; PA13; PA14; PA18; PA21; PA26; PA26; PA28; PA29; PA33; PA39; PA75; PB36; PC20; PC52; PC55 (2 spoons); PC60;

M[20C]-2894: PA11 (2 spoons); PA13; PA21; PA28; PA39; PA39; PA70; PB02; PB02; PB07; PB99 (2 spoons); PC14; PC20; PC49; PC71; PC97; PC98;

M[20C]-2944: PA10; PA11 (2 spoons); PA13; PA14; PA21; PA28; PA39; PA70; PB02; PB07; PB27; PB99; PC11; PC20; PC23; PC67; PC71; PC97; PC98;

M[20C]-2954: PA11; PA12; PA13; PA14; PA21; PA23; PA28; PA33; PA39; PA70; PB01; PB02; PB07; PB99; PC20; PC27; PC52; PC71; PC97; PC98.

TABLE 10-1 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on the extracts of List 10-1 at 1:20 dilution Plank- Biofilm Plank- Biofilm tonic for- tonic for- growth mation growth mation inhibition inhibition inhibition inhibition M[03C]-050 41% 31% M[20C]-2858 58% 32% M[03C]-053 37% 23% M[20C]-2861 58% 31% M[03C]-056 34% 21% M[20C]-2866 57% 43% M[20C]-2808 66% 30% M[20C]-2867 62% 32% M[20C]-2820 54% 27% M[20C]-2868 74% 36% M[20C]-2821 53% 24% M[20C]-2869 85% 45% M[20C]-2826 56% 25% M[20C]-2871 63% 26% M[20C]-2827 51% 26% M[20C]-2876 66% 28% M[20C]-2830 56% 37% M[20C]-2877 81% 43% M[20C]-2831 64% 31% M[20C]-2878 65% 28% M[20C]-2833 80% 39% M[20C]-2879 58% 28% M[20C]-2836 59% 25% M[20C]-2889 62% 41% M[20C]-2837 58% 25% M[20C]-2894 55% 29% M[20C]-2838 70% 30% M[20C]-2944 68% 16% M[20C]-2839 56% 30% M[20C]-2954 75% 27%

List 10-2: extracts from herbal compositions containing Filipendula ulmaria (PA13), Arctostaphylos uva-ursi (PB12) and at least one among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctoria (PA22), Desmodium adscendens (PB07):

M[03C]-051: PA13; PB12; PC20;

M[03C]-054: PA13; PB12; PB07;

M[03C]-057: PA13; PB12; PA22;

M[20C]-2083: PA02; PA13; PA28 (2 spoons); PA31; PA36; PA40; PA79; PA93; PB01; PB12; PB17; PB18; PB31; PB51; PB77; PC20; PC58; PC64; PC66;

M[20C]-2084: PA13; PA28; PA31; PA36; PA61; PA76; PA76; PA79; PA82; PB01; PB10; PB12; PB17; PB31; PB57; PC20; PC36; PC41; PC64; PC66;

M[20C]-2085: PA12; PA13; PA14; PA28; PA30; PA31; PA36; PA79; PA94; PB00; PB01; PB08; PB12; PB17; PB31; PCO2; PC14; PC20; PC64; PC66;

M[20C]-2090: PA08; PA13; PA20; PA23; PA26; PA32; PA46; PA52; PA57; PA79; PA95; PB10; PB12; PB33; PB76; PB82; PC08; PC20; PC21; PD00;

M[20C]-2091: PA08; PA13; PA26; PA52; PA57; PA72; PA79; PA88; PA92; PA95; PB12; PB60; PB76; PB83; PC08; PC11; PC20; PC26; PC68; PD00;

M[20C]-2092: PA08; PA13; PA17; PA26; PA52; PA53; PA57; PA79; PA95; PB06; PB12; PB50; PB76; PC08; PC20; PC20; PC27; PC97; PD00; PD02;

M[20C]-2100: PA13; PA20; PA23; PA32; PA41; PA46; PA53; PA79; PA80; PA90; PB08; PB10; PB12; PB33; PB82; PB85; PC20; PC21; PC28; PC75;

M[20C]-2102: PA13; PA17; PA41; PA53; PA53; PA79; PA80; PA90; PB06; PB08; PB12; PB50; PB85; PC20; PC20; PC27; PC28; PC75; PC97; PD02;

M[20C]-2104: PA13; PA41; PA53; PA61; PA76; PA76; PA79; PA80; PA82; PA90; PB08; PB10; PB12; PB57; PB85; PC20; PC28; PC36; PC41; PC75;

M[20C]-2105: PA12; PA13; PA14; PA30; PA41; PA53; PA79; PA80; PA90; PA94; PB00; PB08; PB08; PB12; PB85; PCO2; PC14; PC20; PC28; PC75;

M[20C]-2110: PA00; PA10; PA13; PA20; PA23; PA25; PA30; PA32; PA46; PA52; PA79; PB05; PB10; PB12; PB33; PB82; PC01; PC20; PC21; PD07;

M[20C]-2111: PA00; PA10; PA13; PA25; PA30; PA52; PA72; PA79; PA88; PA92; PB05; PB12; PB60; PB83; PC01; PC11; PC20; PC26; PC68; PD07;

M[20C]-2120: PA11; PA13; PA16; PA20; PA23; PA32; PA44; PA46; PA60; PA67; PA73; PA79; PB10; PB12; PB33; PB48; PB82; PC17; PC20; PC21;

M[20C]-2122: PA11; PA13; PA16; PA17; PA44; PA53; PA60; PA67; PA73; PA79; PB06; PB12; PB48; PB50; PC17; PC20; PC20; PC27; PC97; PD02;

M[20C]-2131: PA05; PA13; PA18; PA37; PA55; PA72; PA72; PA79; PA88; PA92; PA97; PB04; PB12; PB60; PB83; PC11; PC20; PC26; PC35; PC68;

M[20C]-2135: PA05; PA12; PA13; PA14; PA18; PA30; PA37; PA55; PA72; PA79; PA94; PA97; PB00; PB04; PB08; PB12; PCO2; PC14; PC20; PC35;

M[20C]-2137: PA05; PA13; PA18; PA27; PA37; PA46; PA55; PA72; PA79; PA82; PA97; PB00; PB04; PB11; PB12; PC20; PC35; PC45; PC52; PC67;

M[20C]-2140: PA13; PA14; PA20; PA23; PA32; PA37; PA46; PA79; PA85; PB10; PB12; PB33; PB49; PB82; PB98; PC20; PC21; PC55; PC62; PC98;

M[20C]-2141: PA13; PA14; PA37; PA72; PA79; PA85; PA88; PA92; PB12; PB49; PB60; PB83; PB98; PC11; PC20; PC26; PC55; PC62; PC68; PC98;

M[20C]-2142: PA13; PA14; PA17; PA37; PA53; PA79; PA85; PB06; PB12; PB49; PB50; PB98; PC20; PC20; PC27; PC55; PC62; PC97; PC98; PD02;

M[20C]-2143: PA02; PA13; PA14; PA28; PA37; PA40; PA79; PA85; PA93; PB12; PB18; PB49; PB51; PB77; PB98; PC20; PC55; PC58; PC62; PC98;

M[20C]-2144: PA13; PA14; PA37; PA61; PA76; PA76; PA79; PA82; PA85; PB10; PB12; PB49; PB57; PB98; PC20; PC36; PC41; PC55; PC62; PC98;

M[20C]-2150: PA13; PA20; PA23; PA31; PA32; PA46; PA60; PA74; PA79; PB03; PB10; PB12; PB33; PB82; PC20; PC21; PC29; PC37; PC60; PC86;

M[20C]-2151: PA13; PA31; PA60; PA72; PA74; PA79; PA88; PA92; PB03; PB12; PB60; PB83; PC11; PC20; PC26; PC29; PC37; PC60; PC68; PC86;

M[20C]-2152: PA13; PA17; PA31; PA53; PA60; PA74; PA79; PB03; PB06; PB12; PB50; PC20; PC20; PC27; PC29; PC37; PC60; PC86; PC97; PD02;

M[20C]-2153: PA02; PA13; PA28; PA31; PA40; PA60; PA74; PA79; PA93; PB03; PB12; PB18; PB51; PB77; PC20; PC29; PC37; PC58; PC60; PC86;

M[20C]-2154: PA13; PA31; PA60; PA61; PA74; PA76; PA76; PA79; PA82; PB03; PB10; PB12; PB57; PC20; PC29; PC36; PC37; PC41; PC60; PC86;

M[20C]-2155: PA12; PA13; PA14; PA30; PA31; PA60; PA74; PA79; PA94; PB00; PB03; PB08; PB12; PCO2; PC14; PC20; PC29; PC37; PC60; PC86;

M[20C]-2163: PA06; PA13; PA17; PA20; PA23; PA72; PA79; PA80; PA92; PB01; PB03; PB10; PB12; PB33; PB57; PB82; PC20; PC62; PC86; PC97;

M[20C]-2164: PA13; PA17; PA20; PA41; PA79; PA80; PA86; PA92; PB03; PB10; PB12; PC20; PC26; PC27; PC29; PC55; PC60; PC62; PC86; PC97;

M[20C]-2214: PA13; PA37; PA41; PA79; PA86; PB00; PB10; PB12; PB49; PB82; PC20; PC26; PC27; PC29; PC55; PC60; PC62; PC66; PC86;

M[20C]-2234: PA13; PA41; PA79; PA85; PA86; PB12; PB31; PB50; PB77; PC14; PC20; PC26; PC27; PC29; PC55; PC55; PC60; PC62; PC98;

M[20C]-2241: PA10; PA13; PA14; PA23; PA36; PA37; PA46; PA79; PB00; PB10; PB11; PB12; PB17; PB57; PC20; PC27; PC29; PC37; PC64; PC68;

M[20C]-2242: PA13; PA14; PA17; PA23; PA36; PA37; PA60; PA61; PA76; PA79; PA82; PB00; PB06; PB12; PB49; PC20; PC27; PC29; PC68; PC98;

M[20C]-2243: PA13; PA14; PA23; PA36; PA37; PA52; PA53; PA73; PA79; PA92; PB00; PB01; PB12; PB18; PB60; PC20; PC20; PC27; PC29; PC68;

M[20C]-2248: PA13; PA14 (2 spoons); PA23; PA25; PA36; PA36; PA37; PA46; PA79; PA85; PB00; PB12; PCO2; PC20; PC27; PC29; PC29; PC55; PC68;

M[20C]-2249: PA10; PA13; PA14; PA23; PA36; PA37; PA53; PA74; PA79; PA92; PB00; PB12; PC20; PC27; PC29; PC62; PC67; PC68; PC86; PC97;

M[20C]-2250: PA13; PA23; PA37; PA76; PA79; PB10; PB12; PB33; PB50; PB82; PB82; PB85; PB98; PC01; PC11; PC20; PC45; PC55; PC60; PD02;

M[20C]-2253: PA13; PA37; PA52; PA53; PA73; PA79; PA92; PB01; PB10; PB12; PB18; PB33; PB50; PB60; PB82; PB85; PC01; PC20; PC20; PC45;

M[20C]-2254: PA11; PA13; PA17; PA27; PA37; PA79; PB00; PB10; PB12; PB33; PB50; PB82; PB83; PB85; PB92; PB98; PC01; PC20; PC27; PC45;

M[20C]-2255: PA13; PA36; PA37; PA53; PA72; PA79; PA80; PB03; PB10; PB12; PB33; PB49; PB50; PB82; PB82; PB85; PC01; PC20; PC45; PC68;

M[20C]-2256: PA13; PA23; PA37; PA53; PA61; PA79; PA85; PB10; PB12; PB33; PB50; PB60; PB82; PB85; PC01; PC11; PC20; PC29; PC45; PD02;

M[20C]-2257: PA13; PA37; PA37; PA60; PA72; PA79; PB10; PB12; PB33; PB49; PB50; PB82; PB85; PC01; PC08; PC20; PC45; PC68; PC98; PD02;

M[20C]-2258: PA13; PA14; PA25; PA36; PA37; PA46; PA79; PA85; PB10; PB12; PB33; PB50; PB82; PB85; PC01; PCO2; PC20; PC29; PC45; PC55;

M[20C]-2259: PA10; PA13; PA37; PA53; PA74; PA79; PA92; PB10; PB12; PB33; PB50; PB82; PB85; PC01; PC20; PC45; PC62; PC67; PC86; PC97;

M[20C]-2261: PA10; PA13; PA23; PA36; PA37; PA46; PA53; PA73; PA79; PA97; PB10; PB11; PB12; PB17; PB57; PC20; PC26; PC29; PC37; PC64;

M[20C]-2278: PA10; PA13; PA14; PA25; PA28; PA30; PA36; PA37; PA41; PA46; PA79; PA85; PB12; PB50; PB60; PCO2; PC20; PC29; PC55; PC66;

M[20C]-2281: PA10; PA13; PA17; PA46; PA52; PA79; PA82; PA92; PA96; PB10; PB11; PB12; PB17; PB57; PB98; PC20; PC36; PC37; PC64; PC86;

M[20C]-2321: PA13; PA46; PA53; PA79; PA82; PA92; PB03; PB10; PB11; PB12; PB83; PB85; PC20; PC20; PC21; PC55; PC60; PC86; PC97; PD02;

M[20C]-2406: PA13; PA37; PA46; PA53; PA79; PA85; PB01; PB05; PB10; PB11; PB12; PB17; PB49; PB98; PC20; PC36; PC37; PC45; PC58; PC67;

M[20C]-2420: PA10; PA13; PA14; PA36; PA46; PA79; PB10; PB10; PB11; PB11; PB12; PB17; PB17; PB33; PB82; PC20; PC54; PC55; PC68; PC98;

M[20C]-2441: PA00; PA13; PA14; PA17; PA17; PA37; PA37; PA46; PA74; PA79; PB00; PB03; PB10; PB12; PB49; PC08; PC20; PC35; PC62; PC86;

M[20C]-2443: PA00; PA13; PA14; PA14; PA17; PA36; PA37; PA79; PB00; PB01; PB10; PB12; PCO2; PC08; PC11; PC20; PC29; PC62; PC67; PC86;

M[20C]-2449: PA00; PA13; PA14; PA17; PA36; PA37; PA60; PA79; PA85; PB00; PB10; PB12; PB82; PB98; PC08; PC20; PC29; PC62; PC86; PC98;

M[20C]-2451: PA10; PA13; PA17; PA23; PA37; PA46; PA74; PA76; PA79; PB00; PB03; PB08; PB10; PB12; PB49; PB49; PC20; PC35; PC97; PC98;

M[20C]-2460: PA08; PA13; PA14; PA36; PA36; PA46; PA79; PB10; PB11; PB12; PB17; PB17; PB98; PC20; PC36; PC54; PC62; PC68; PC68; PC86;

M[20C]-2488: PA10; PA13; PA37; PA46; PA53; PA74; PA79; PA82; PB00; PB00; PB12; PB49; PC20; PC29; PC37; PC45; PC54; PC74; PC86; PC98;

M[20C]-2545: PA00; PA08; PA10; PA13; PA46; PA73; PA76; PA79; PA90; PB01; PB08; PB10; PB12; PB17; PB33; PB33; PC20; PC55; PC68; PD02;

M[20C]-2551: PA13; PA14; PA16; PA27; PA36; PA79; PA82; PA92; PA95; PB10; PB12; PB57; PB85; PC11; PC20; PC29; PC29; PC54; PC64; PC98;

M[20C]-2553: PA13; PA16; PA23; PA60; PA79; PA82; PA95; PB00; PB10; PB10; PB12; PB33; PB49; PB50; PB85; PCO2; PC11; PC20; PC29; PC98;

M[20C]-2555: PA00; PA10; PA13; PA16; PA79; PA82; PA90; PA95; PB08; PB10; PB10; PB12; PB33; PB85; PC11; PC20; PC29; PC68; PC98; PD02;

M[20C]-2580: PA10; PA13; PA25; PA37; PA40; PA46; PA79; PB11; PB12; PB33; PB49; PB82; PB85; PB98; PC20; PC29; PC29; PC36; PC45; PC97;

M[20C]-2589: PA13; PA14; PA17; PA40; PA46; PA79; PA80; PB11; PB12; PB33; PB49; PB85; PB98; PC20; PC26; PC29; PC37; PC86; PC97; PD02;

M[20C]-2622: PA13; PA25; PA36; PA36; PA37; PA46; PA46; PA79; PA82; PB12; PB49; PB82; PC20; PC20; PC54; PC55; PC64; PC68; PC86; PC98;

M[20C]-2627: PA13; PA14; PA36; PA46; PA76; PA79; PA80; PA96; PB12; PB49; PB49; PC20; PC20; PC45; PC54; PC55; PC64; PC97; PC98; PC98;

M[20C]-2641: PA05; PA13; PA16; PA27; PA36; PA37; PA41; PA79; PA96; PB05; PB10; PB12; PB82; PB85; PB98; PC11; PC20; PC54; PC68; PD02;

M[20C]-2651: PA13; PA36; PA41; PA46; PA79; PA92; PB00; PB01; PB05; PB12; PB33; PB82; PB82; PB85; PB98; PC20; PC54; PC55; PC86; PD02;

M[20C]-2654: PA13; PA36; PA46; PA79; PA92; PB00; PB00; PB01; PB08; PB11; PB12; PB33; PB82; PC01; PC20; PC55; PC55; PC64; PC86; PC86;

M[20C]-2717: PA13; PA14; PA46; PA79; PB00; PB10; PB12; PB49; PB50; PB77; PB85; PB85; PCO2; PC20; PC29; PC64; PC64; PC68; PC98; PD02;

M[20C]-2728: PA05; PA13; PA16; PA30; PA46; PA60; PA61; PA79; PA85; PB04; PB10; PB12; PB49; PB82; PB82; PB85; PB98; PC11; PC20; PC37;

M[20C]-2730: PA13; PA36; PA37; PA46; PA61; PA79; PA82; PA85; PB00; PB12; PB17; PB33; PB82; PB85; PC01; PCO2; PC20; PC29; PC64; PC98;

M[20C]-2738: PA05; PA13; PA37; PA46; PA61; PA61; PA79; PA85; PB10; PB12; PB82; PB82; PB85; PB85; PB98; PCO2; PC20; PC29; PC37; PC64;

M[20C]-2788: PA00; PA05; PA13; PA27; PA46; PA61; PA79; PA82; PB00; PB10; PB12; PB82; PB85; PB85; PB98; PC20; PC37; PC37; PC58; PC97;

M[20C]-2805: PA11; PA13; PA13; PA14; PA22; PA23; PA28; PA33; PA35; PA69; PB07; PB12; PB38; PB88; PB88; PC20; PC24; PC55; PC55; PC60;

M[20C]-2812: PA11; PA11; PA13; PA20; PA23; PA29; PA30; PA83; PA98; PB02; PB12; PB27; PB38; PC11; PC11; PC20; PC23; PC37; PC51; PC60;

M[20C]-2815: PA11; PA13; PA14; PA22; PA23; PA29; PA33; PA35; PA98; PB02; PB12; PB27; PB38; PB38; PB88; PC11; PC20; PC23; PC37; PC55;

M[20C]-2840: PA10; PA11; PA12 (2 spoons); PA13; PA14; PA18; PA28; PA33; PA67; PA67; PB07 (2 spoons); PB12; PB60; PC23; PC35; PC37; PC55; PC67;

M[20C]-2841: PA12; PA13 (2 spoons); PA19; PA22; PA28; PA33; PA67; PB07 (2 spoons); PB12; PB36; PC20; PC23; PC33; PC37; PC49; PC55; PC67; PC97;

M[20C]-2842: PA11; PA12; PA13 (2 spoons); PA20; PA28; PA30; PA33; PA67; PA83; PB07; PB12 (2 spoons); PC11; PC20; PC23; PC37; PC51; PC60; PC67;

M[20C]-2843: PA10; PA11; PA12; PA13; PA14; PA24; PA26; PA28; PA33; PA67; PB07; PB12; PB33; PB88; PC12; PC23; PC23; PC27; PC37; PC67;

M[20C]-2844: PA12; PA13; PA13; PA15; PA18; PA20; PA28; PA32; PA33; PA67 (2 spoons); PB07; PB12; PB12; PC20; PC23; PC37; PC49; PC55; PC67;

M[20C]-2845: PA12; PA13 (2 spoons); PA14; PA22; PA28; PA33; PA33; PA35; PA67; PB07; PB12 (2 spoons); PB38; PB88; PC20; PC23; PC37; PC55; PC67;

M[20C]-2847: PA10; PA12; PA13 (2 spoons); PA26; PA28; PA33; PA57; PA67; PB02; PB07; PB07; PB12; PB28; PC11; PC23; PC23; PC37; PC49; PC67;

M[20C]-2849: PA12; PA13 (2 spoons); PA14; PA25; PA26; PA28; PA33; PA39; PA52; PA57; PA67; PB07; PB12; PB33; PC23; PC37; PC67; PC71; PC97;

M[20C]-2852: PA11; PA13; PA20 (2 spoons); PA23; PA24; PA30; PA67; PA83; PB07; PB12; PB28; PB33; PC11; PC12; PC20; PC20; PC21; PC51; PC60;

M[20C]-2854: PA13; PA15; PA18; PA20 (2 spoons); PA23; PA24; PA32; PA67 (2 spoons); PB07; PB12; PB28; PB33; PC12; PC20 (2 spoons); PC21; PC49; PC55;

M[20C]-2855: PA13; PA14; PA20; PA22; PA23; PA24; PA33; PA35; PA67; PB07; PB12; PB28; PB33; PB38; PB88; PC12; PC20; PC21; PC55;

M[20C]-2893: PA00; PA06; PA11; PA12; PA13; PA23; PA26; PA28; PA33; PA39; PB02; PB07 (2 spoons); PB12; PB99; PC14; PC20; PC49; PC98; PD02;

M[20C]-2897: PA11 (2 spoons); PA13; PA14; PA24; PA29; PA39; PA83; PB02; PB07; PB12; PB60; PB99; PC11; PC14; PC20; PC20; PC49; PC51; PC98;

M[20C]-2930: PA11; PA13; PA14; PA23; PA26; PA28; PA33; PA35; PA83; PB12; PB28; PB33; PB38; PB77; PB88; PC20; PC23; PC37; PC60; PC60;

M[20C]-2933: PA00; PA06; PA11; PA12; PA13; PA14; PA23; PA26; PA28 (2 spoons); PA33; PA33; PB07; PB12; PB12; PB28; PB38; PB77; PC60; PD02;

M[20C]-2935: PA10; PA11; PA13; PA14; PA28; PA29; PA33; PA57; PA67; PB07; PB12; PB28 (2 spoons); PB38; PB77; PB77; PC11; PC20; PC52; PC60;

M[20C]-2936: PA11 (2 spoons); PA13; PA14; PA18; PA28; PA33; PA57; PA67; PB02; PB12; PB28; PB38 (2 spoons); PC11; PC20; PC27; PC52; PC60;

M[20C]-2937: PA11; PA11; PA13; PA14 (3 spoons); PA28; PA29; PA33; PA83; PB12 (2 spoons); PB28; PB38; PB60; PB77; PC11; PC20; PC51; PC60;

M[20C]-2939: PA10; PA11; PA12; PA13 (2 spoons); PA14; PA18; PA26; PA28; PA33 (2 spoons); PA39; PB12; PB28; PB38; PB77; PC20; PC52; PC55; PC60;

M[20C]-2943: PA00; PA06; PA10; PA11; PA12; PA13; PA14; PA23; PA26; PA28; PA33; PB07 (2 spoons); PB12; PB27; PC11; PC20; PC23; PC67; PD02;

M[20C]-2947: PA10; PA11 (2 spoons); PA13; PA14 (2 spoons); PA24; PA29; PA83; PB07; PB12; PB27; PB60; PC11 (2 spoons); PC20 (2 spoons); PC23; PC51; PC67;

M[20C]-2953: PA00; PA06; PA12 (2 spoons); PA13; PA14; PA23 (2 spoons); PA26; PA28; PA33 (2 spoons); PB01; PB07 (2 spoons); PB12; PC20; PC27; PC52; PD02;

M[20C]-2957: PA11; PA12; PA13; PA14 (2 spoons); PA23; PA24; PA29; PA33; PA83; PB01; PB07; PB12; PB60; PC11; PC20 (2 spoons); PC27; PC51; PC52.

TABLE 10-2 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on the mixes of List 10-2 at 1:20 dilution Plank- Biofilm Plank- Biofilm tonic for- tonic for- growth mation growth mation inhi- inhi- inhi- inhi- bition bition bition bition M[03C]-051 37% 24% M[20C]-2321 55% 30% M[03C]-054 30% 13% M[20C]-2406 58% 31% M[03C]-057 27% 22% M[20C]-2420 55% 34% M[20C]-2083 84% 55% M[20C]-2441 52% 36% M[20C]-2084 72% 40% M[20C]-2443 58% 33% M[20C]-2085 70% 45% M[20C]-2449 55% 32% M[20C]-2090 57% 46% M[20C]-2451 53% 28% M[20C]-2091 66% 35% M[20C]-2460 54% 37% M[20C]-2092 71% 43% M[20C]-2488 54% 27% M[20C]-2100 56% 40% M[20C]-2545 52% 29% M[20C]-2102 53% 41% M[20C]-2551 52% 29% M[20C]-2104 60% 43% M[20C]-2553 52% 27% M[20C]-2105 81% 55% M[20C]-2555 58% 39% M[20C]-2110 59% 43% M[20C]-2580 58% 44% M[20C]-2111 63% 46% M[20C]-2589 56% 35% M[20C]-2120 66% 28% M[20C]-2622 57% 40% M[20C]-2122 72% 37% M[20C]-2627 58% 39% M[20C]-2131 54% 37% M[20C]-2641 53% 36% M[20C]-2135 59% 40% M[20C]-2651 53% 33% M[20C]-2137 78% 50% M[20C]-2654 53% 35% M[20C]-2140 60% 35% M[20C]-2717 55% 30% M[20C]-2141 56% 40% M[20C]-2728 56% 24% M[20C]-2142 61% 34% M[20C]-2730 55% 27% M[20C]-2143 77% 37% M[20C]-2738 52% 26% M[20C]-2144 74% 39% M[20C]-2788 56% 25% M[20C]-2150 65% 27% M[20C]-2805 71% 25% M[20C]-2151 61% 34% M[20C]-2812 54% 31% M[20C]-2152 66% 32% M[20C]-2815 52% 29% M[20C]-2153 82% 50% M[20C]-2840 55% 31% M[20C]-2154 67% 26% M[20C]-2841 51% 31% M[20C]-2155 63% 32% M[20C]-2842 53% 35% M[20C]-2163 61% 40% M[20C]-2843 58% 34% M[20C]-2164 70% 35% M[20C]-2844 76% 42% M[20C]-2214 76% 37% M[20C]-2845 46% 33% M[20C]-2234 56% 31% M[20C]-2847 61% 41% M[20C]-2241 60% 40% M[20C]-2849 76% 32% M[20C]-2242 59% 33% M[20C]-2852 77% 32% M[20C]-2243 63% 38% M[20C]-2854 57% 27% M[20C]-2248 84% 49% M[20C]-2855 66% 35% M[20C]-2249 63% 36% M[20C]-2893 61% 37% M[20C]-2250 58% 36% M[20C]-2897 66% 36% M[20C]-2253 76% 37% M[20C]-2930 81% 52% M[20C]-2254 55% 25% M[20C]-2933 60% 32% M[20C]-2255 60% 29% M[20C]-2935 65% 34% M[20C]-2256 58% 30% M[20C]-2936 68% 41% M[20C]-2257 59% 25% M[20C]-2937 78% 41% M[20C]-2258 81% 39% M[20C]-2939 77% 44% M[20C]-2259 60% 29% M[20C]-2943 67% 29% M[20C]-2261 52% 25% M[20C]-2947 71% 40% M[20C]-2278 53% 25% M[20C]-2953 66% 38% M[20C]-2281 56% 26% M[20C]-2957 71% 45%

List 10-3: herbal compositions of processed samples containing: Camellia sinensis (PA21), Arctostaphylos uva-ursi (PB12) and at least one among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctorial (PA22), Desmodium adscendens (PB07):

M[03C]-052: PA21; P1B12; PC20;

M[03C]-055: PA21; P1B12; P1B07;

M[03C]-058: PA21; PB12; PA22;

M[20C]-2888: PA14; PA15; PA18; PA21; PA21; PA23; PA26; PA28; PA28; PA29; PA39; PA57; PA75; PB07; PB12; PB36; PC11; PC55; PC55; PC60;

M[20C]-2913: PA00; PA06; PA12; PA21; PA23; PA26; PA26; PA27; PA28; PA33; PB07; PB07; PB12 (2 spoons); PB33; PC20; PC24; PC41; PC71; PD02;

M[20C]-2918: PA15; PA18; PA21 (2 spoons); PA23; PA26; PA27; PA28; PA57; PB07 (2 spoons); PB12 (2 spoons); PB33; PC11; PC20; PC24; PC41; PC55; PC71;

M[20C]-2920: PA20; PA21; PA23; PA26; PA28; PA35; PA35; PA67; PA83; PB12; PB33; PB88; PC12; PC20; PC23; PC37; PC37; PC60 (2 spoons); PC74;

M[20C]-2923: PA00; PA06; PA12; PA20; PA21; PA23; PA26; PA28 (2 spoons); PA33; PA35; PA67; PB07; PB12 (2 spoons); PC12; PC37; PC60; PC74; PD02;

M[20C]-2928: PA15; PA18; PA20; PA21 (2 spoons); PA23; PA28 (2 spoons); PA35; PA57; PA67; PB07; PB12 (2 spoons); PC11; PC12; PC37; PC55; PC60; PC74;

M[20C]-2887: PA11; PA14; PA14; PA21; PA24; PA26; PA28; PA29 (2 spoons); PA39; PA75; PA83; PB12; PB36; PB60; PC11; PC20; PC51; PC55; PC60;

M[20C]-2914: PA11; PA21 (2 spoons); PA26; PA27; PA28; PA39; PA70; PB02; PB07; PB12; PB33; PB99; PC20; PC24; PC41; PC71 (2 spoons); PC97; PC98;

M[20C]-2915: PA10; PA21; PA26; PA27; PA29; PA57; PA67; PB07 (2 spoons); PB12; PB28; PB33; PB77; PC11; PC20 (2 spoons); PC24; PC41; PC52; PC71;

M[20C]-2916: PA11; PA18; PA21; PA26; PA27; PA57; PA67; PB02; PB07; PB12; PB33; PB38; PC11; PC20; PC20; PC24; PC27; PC41; PC52; PC71;

M[20C]-2917: PA11; PA14; PA21; PA24; PA26; PA27; PA29; PA83; PB07; PB12 (2 spoons); PB33; PB60; PC11; PC20 (2 spoons); PC24; PC41; PC51; PC71;

M[20C]-2925: PA10; PA20; PA21; PA28; PA29; PA35; PA57; PA67 (2 spoons); PB07; PB12; PB28; PB77; PC11; PC12; PC20; PC37; PC52; PC60; PC74;

M[20C]-2926: PA11; PA18; PA20; PA21; PA28; PA35; PA57; PA67 (2 spoons); PB02; PB12; PB38; PC11; PC12; PC20; PC27; PC37; PC52; PC60; PC74;

M[20C]-2927: PA11; PA14; PA20; PA21; PA24; PA28; PA29; PA35; PA67; PA83; PB12; PB12; PB60; PC11; PC12; PC20; PC37; PC51; PC60; PC74;

M[20C]-2910: PA21; PA23; PA26 (2 spoons); PA27; PA35; PA83; PB07; PB12; PB33 (2 spoons); PB88; PC20 (2 spoons); PC23; PC24; PC37; PC41; PC60; PC71.

TABLE 10-3 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on the mixes of List 10-2 at 1:20 dilution Plank- Biofilm Plank- Biofilm tonic for- tonic for- growth mation growth mation inhibition inhibition inhibition inhibition M[03C]-052 31% 13% M[20C]-2887 46% 8% M[03C]-055 29% 10% M[20C]-2914 29% 38% M[03C]-058 31% 18% M[20C]-2915 30% 35% M[20C]-2888 48% 27% M[20C]-2916 39% 40% M[20C]-2913 46% 37% M[20C]-2917 40% 41% M[20C]-2918 46% 32% M[20C]-2925 41% 36% M[20C]-2920 54% 30% M[20C]-2926 47% 36% M[20C]-2923 51% 28% M[20C]-2927 41% 41% M[20C]-2928 46% 32% M[20C]-2910 46% 39%

List 10-4: extracts from herbal compositions containing: Filipendula ulmaria (PA13), Camellia sinensis (PA21), Arctostaphylos uva-ursi (PB12) and at least one among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctorial (PA22), Desmodium adscendens (PB07):

M[04C]-025: PA13; PA21; PB12; PC20;

M[04C]-026: PA13; PA21; PB12; PB07;

M[04C]-027: PA13; PA21; PB12; PA22;

M[20C]-2898: PA11; PA13; PA15; PA18; PA21; PA23; PA28; PA39; PA57; PB02; PB07 (2 spoons); PB07; PB12; PB99; PC11; PC14; PC20; PC49; PC55; PC98;

M[20C]-2900: PA10; PA11; PA13; PA21; PA23; PA25; PA26; PA35; PA69; PA83; PB12; PB32; PB33; PB88; PC20 (2 spoons); PC23; PC37; PC55; PC60;

M[20C]-2903: PA00; PA06; PA10; PA11; PA12; PA13; PA21; PA23; PA25; PA26; PA28; PA33; PA69; PB07; PB12 (2 spoons); PB32; PC20; PC55; PD02;

M[20C]-2908: PA10; PA11; PA13; PA15; PA18; PA21 (2 spoons); PA23; PA25; PA28; PA57; PA69; PB07; PB12 (2 spoons); PB32; PC11; PC20; PC55 (2 spoons);

M[20C]-2938: PA11; PA13; PA14; PA15; PA18; PA21; PA23; PA28 (2 spoons); PA33; PA57; PB07; PB12 (2 spoons); PB28; PB38; PB77; PC11; PC55; PC60;

M[20C]-2948: PA10; PA11; PA13; PA14; PA15; PA18; PA21; PA23; PA28; PA57; PB07 (2 spoons); PB12; PB27; PC11 (2 spoons); PC20; PC23; PC55; PC67;

M[20C]-2951: PA12; PA13 (2 spoons); PA14; PA21; PA23; PA24; PA33; PA39; PA67; PB01; PB07; PB12; PB77; PC20; PC27; PC33; PC49; PC52; PC60;

M[20C]-2952: PA12; PA13 (2 spoons); PA14; PA21; PA23; PA25; PA26; PA33; PA37; PA67; PB01; PB07 (2 spoons); PB12; PC20; PC27; PC49; PC52; PC71;

M[20C]-2958: PA12; PA13; PA14; PA15; PA18; PA21; PA23 (2 spoons); PA28; PA33; PA57; PB01; PB07 (2 spoons); PB12; PC11; PC20; PC27; PC52; PC55;

M[20C]-2822: PA11 (2 spoons); PA13 (2 spoons); PA20 (2 spoons); PA21; PA25; PA29; PA30; PA33; PA35; PA75; PA83; PB12; PC11; PC20; PC33; PC51; PC60;

M[20C]-2824: PA11; PA13 (2 spoons); PA15; PA18; PA20 (2 spoons); PA21; PA25; PA29; PA32; PA33; PA35; PA67; PA75; PB12; PC20; PC33; PC49; PC55;

M[20C]-2825: PA11; PA13 (2 spoons); PA14; PA20; PA21; PA22; PA25; PA29; PA33 (2 spoons); PA35 (2 spoons); PA75; PB12; PB38; PB88; PC20; PC33; PC55;

M[20C]-2834: PA13 (2 spoons); PA15; PA18; PA20; PA21; PA22; PA25; PA29; PA32 (2 spoons); PA34; PA67; PB12; PB88; PC20; PC33; PC49; PC55; PC98;

M[20C]-2835: PA13 (2 spoons); PA14; PA21; PA22 (2 spoons); PA25; PA29; PA32; PA33; PA34; PA35; PB12; PB38; PB88 (2 spoons); PC20; PC33; PC55; PC98;

M[20C]-2848: PA12 (2 spoons); PA13 (2 spoons); PA18; PA21; PA24; PA28; PA33; PA67; PB07; PB12; PC23 (2 spoons); PC37; PC49; PC52; PC55; PC67; PC97;

M[20C]-2862: PA11; PA13; PA20; PA21; PA22; PA30; PA83; PB12; PB28; PB60; PC11 (2 spoons); PC20 (2 spoons); PC23; PC51; PC55; PC60 (2 spoons); PC74;

M[20C]-2864: PA13; PA15; PA18; PA20; PA21; PA22; PA32; PA67; PB12; PB28; PB60; PC11; PC20 (2 spoons); PC23; PC49; PC55 (2 spoons); PC60; PC74;

M[20C]-2865: PA13; PA14; PA21; PA22 (2 spoons); PA33; PA35; PB12; PB28; PB38; PB60; PB88; PC11; PC20 (2 spoons); PC23; PC55 (2 spoons); PC60; PC74;

M[20C]-2872: PA10; PA11 (2 spoons); PA13; PA20; PA21; PA30; PA35; PA57; PA83; PB07; PB12; PB77; PC11; PC20 (2 spoons); PC23; PC51; PC60; PC71;

M[20C]-2874: PA10; PA11; PA13; PA15; PA18; PA20; PA21; PA32; PA35; PA57; PA67; PB07; PB12; PB77; PC20 (2 spoons); PC23; PC49; PC55; PC71;

M[20C]-2875: A10; A11; A13; A14; A21; A22; A33; A35; A35; A57; B07; B12; B38; B77; B88; C20 (2 spoons); C23; C55; C71;

M[20C]-2882: PA13; PA14; PA21 (2 spoons); PA25; PA26 (2 spoons); PA28; PA29; PA37; PA39; PA67; PA75; PB07; PB12; PB36; PC49; PC55; PC60; PC71;

M[20C]-2891: PA11; PA13 (2 spoons); PA21; PA24; PA39; PA39; PA67; PB02; PB07; PB12; PB77; PB99; PC14; PC20; PC33; PC49 (2 spoons); PC60; PC98;

M[20C]-2892: PA11; PA13 (2 spoons); PA21; PA25; PA26; PA37; PA39; PA67; PB02; PB07 (2 spoons); PB12; PB99; PC14; PC20; PC49 (2 spoons); PC71; PC98;

M[20C]-2901: PA10; PA11; PA13 (2 spoons); PA21 (2 spoons); PA24; PA25; PA39; PA67; PA69; PB12 (2 spoons); PB32; PB77; PC20; PC33; PC49; PC55; PC60;

M[20C]-2902: PA10; PA11; PA13 (2 spoons); PA21 (2 spoons); PA25 (2 spoons); PA26; PA37; PA67; PA69; PB07; PB12 (2 spoons); PB32; PC20; PC49; PC55; PC71;

M[20C]-2904: PA10; PA11 (2 spoons); PA13; PA21 (2 spoons); PA25; PA28; PA39; PA69; PA70; PB02; PB12; PB32; PB99; PC20; PC55; PC71; PC97; PC98;

M[20C]-2905: PA10 (2 spoons); PA11; PA13; PA21; PA25; PA29; PA57; PA67; PA69; PB07; PB12; PB28; PB32; PB77; PC11; PC20 (2 spoons); PC52; PC55;

M[20C]-2906: PA10; PA11 (2 spoons); PA13; PA18; PA21; PA25; PA57; PA67; PA69; PB02; PB12; PB32; PB38; PC11; PC20 (2 spoons); PC27; PC52; PC55;

M[20C]-2907: PA10; PA11 (2 spoons); PA13; PA14; PA21; PA24; PA25; PA29; PA69; PA83; PB12 (2 spoons); PB32; PB60; PC11; PC20 (2 spoons); PC51; PC55;

M[20C]-2909: A10 (2 spoons); A11; A12; A13 (2 spoons); A18; A21; A25; A26; A33; A39; A69; B12; B32; C20 (2 spoons); C52; C55 (2 spoons);

M[20C]-2911: PA13; PA21 (2 spoons); PA24; PA26; PA27; PA39; PA67; PB07; PB12 (2 spoons); PB33; PB77; PC20; PC24; PC33; PC41; PC49; PC60; PC71;

M[20C]-2912: PA13; A21 (2 spoons); PA25; PA26 (2 spoons); PA27; PA37; PA67; PB07 (2 spoons); PB12 (2 spoons); PB33; PC20; PC24; PC41; PC49; PC71 (2 spoons);

M[20C]-2919: PA10; PA12; PA13; PA18; PA21; PA26 (2 spoons); PA27; PA33; PA39; PB07; PB12; PB33; PC20 (2 spoons); PC24; PC41; PC52; PC55; PC71;

M[20C]-2922: PA13; PA20; PA21 (2 spoons); PA25; PA26; PA28; PA35; PA37; PA67 (2 spoons); PB07; PB12 (2 spoons); PC12; PC37; PC49; PC60; PC71; PC74;

M[20C]-2929: PA10; PA12; PA13; PA18; PA20; PA21; PA26; PA28; PA33; PA35; PA39; PA67; PB12; PC12; PC20; PC37; PC52; PC55; PC60; PC74;

M[20C]-2932: PA11; PA13 (2 spoons); PA14; PA21; PA25; PA26; PA28; PA33; PA37; PA67; PB07; PB12 (2 spoons); PB28; PB38; PB77; PC49; PC60; PC71;

M[20C]-2941: PA10; PA11; PA13 (2 spoons); PA14; PA21; PA24; PA39; PA67; PB07; PB12; PB27; PB77; PC11; PC20; PC23; PC33; PC49; PC60; PC67;

M[20C]-2942: PA10; PA11; PA13 (2 spoons); PA14; PA21; PA25; PA26; PA37; PA67; PB07 (2 spoons); PB12; PB27; PC11; PC20; PC23; PC49; PC67; PC71;

TABLE 10-4 average of planktonic growth inhibition percentage and biofilm formation inhibition percentage efficacy on the 6 tested Staphylococcus aureus strains of the processed sample based on the mixes of List 10-4 at 1:20 dilution Bio- Bio- Plank- film Plank- film tonic for- tonic for- growth mation growth mation inhi- inhi- inhi- inhi- bition bition bition bition M[04C]-025 57% 18% M[20C]-2872 59% 36% M[04C]-026 48% 16% M[20C]-2874 78% 39% M[04C]-027 49% 18% M[20C]-2875 83% 47% M[20C]-2898 60% 21% M[20C]-2882 61% 28% M[20C]-2900 78% 32% M[20C]-2891 70% 27% M[20C]-2903 48% 28% M[20C]-2892 76% 39% M[20C]-2908 54% 15% M[20C]-2901 59% 29% M[20C]-2938 70% 26% M[20C]-2902 51% 30% M[20C]-2948 56% 22% M[20C]-2904 62% 24% M[20C]-2951 47% 17% M[20C]-2905 55% 37% M[20C]-2952 60% 19% M[20C]-2906 59% 37% M[20C]-2958 53% 31% M[20C]-2907 75% 41% M[20C]-2822 46% 38% M[20C]-2909 82% 49% M[20C]-2824 60% 32% M[20C]-2911 55% 30% M[20C]-2825 76% 38% M[20C]-2912 59% 29% M[20C]-2834 55% 27% M[20C]-2919 75% 48% M[20C]-2835 64% 29% M[20C]-2922 63% 32% M[20C]-2848 52% 29% M[20C]-2929 54% 30% M[20C]-2862 46% 31% M[20C]-2932 73% 39% M[20C]-2864 51% 29% M[20C]-2941 63% 48% M[20C]-2865 56% 43% M[20C]-2942 56% 34%

Comparative Example 4

As a comparative Example, we report several extracts of herbal compositions containing only 1 plant among 3: Filipendula ulmaria (PA13), Camellia sinensis (PA21), Arctostaphylos uva-ursi (PB12) and only 1 among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctoria (PA22), Desmodium adscendens (PB07).

5 herbal compositions having the content reported in List 10-5 were prepared according to the previously described Method 1.

List 10-5 extracts from herbal compositions containing the following plants

M[20P]-318: PA15; PA21; PA34; PA35; PA82; PA83; PA85; PA92; PA96; PB03; PB07; PB26; PB30; PB36; PB79; PC11; PC49; PC91; PC92; PD10;

M[20P]-329: PA10; PA28; PA34; PA36; PASS; PA72; PA83; PA85; PA91; PB03; PB12; PB16; PB28; PB35; PB49; PB92; PC20; PC23; PC64; PC85;

M[20P]-333: PA04; PA05; PA13; PA16; PA22; PA39; PA51; PA55; PA57; PA72; PA82; PA92; PA98; PB05; PB20; PB36; PB43; PB74; PB77; PC37;

M[20P]-362: PA21; PA23; PA25; PA33; PA37; PA57; PA69; PA81; PA92; PB16; PB25; PB26; PB28; PB29; PB78; PC20; PC21; PC71; PC83; PD02;

M[20P]-513: PA12; PA14; PA14; PA20; PA20; PA21; PA22; PA33; PA35; PA52; PA57; PA69; PB00; PB32; PB38; PC24; PC37; PC52; PC55; PC67;

Herbal compositions containing 20 plant powders were prepared according to the previously described Method A.

Starting from the herbal compositions, corresponding water extracts (or processed samples) were prepared according to the previously described Method B using 40 mL of water containing 100 g/l of sucrose for water extraction and including the decantation step before filtration.

The biological activity of each processed sample was determined according to the previously described Method C by using the following Staphylococcus aureus strains: ATCC 25923, ATCC 49476, ATCC 6538, ATCC 51740, ATCC 29213 and ATCC 14775, with the 1:20 final dilutions of processed samples.

The bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from the herbal compositions of List 10-4 is given in Table 10-5. Each percentage is the average of 2 measurements of the same condition in two different wells. Negative percentages reflect growth promotion and biofilm promotion, instead of the desired inhibition. It is appreciated that such extracts give very poor, if any inhibition as compared to the extracts of Example 10.

TABLE 10-5 bacteria planktonic growth inhibition percentage average and biofilm formation inhibition percentage average over all the tested strains of the processed samples obtained from the herbal compositions of List 10-4 Planktonic growth Biofilm formation inhibition inhibition M[20C]-318 −10%  0% M[20C]-329 12% 9% M[20C]-333  3% 18%  M[20C]-362 22% 3% M[20C]-513 −21%  21% 

Example 11

In this Example, we illustrate antimicrobial activities against 14 different microbial species described in Table11-1 of 10 mixtures extracts obtained from the herbal compositions disclosed in List 11-1 comprising at least 2 plants among 3: Filipendula ulmaria (PA13), Camellia sinensis (PA21), Arctostaphylos uva-ursi (PB12) and at least 1 plant among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctorial (PA22), and Desmodium adscendens (PB07).

TABLE 11-1 microbial species and strains used in Example 11 Innoculum Strain concentration Strain full name code (bacteria/mL) Staphylococcus aureus subsp. aureus B-A49 2.105.5 (MRSA) ATCC ® 33592 ™ Staphylococcus epidermidis ATCC ® 14990 ™ B-A08 2.105    Staphylococcus pseudintermedius B-A09 2.105    ATCC ® 49444 ™ Candida albicans ATCC ® 60193 ™ B-A61 2.106.5 Escherichia coli ATCC ® 11775 ™ B-A67 2.105.5 Klebsiella pneumoniae ATCC ® 13883 ™ B-A68 2.105.5 Listeria innocua ATCC ® 33090 ™ B-A69 2.105.5 Listeria monocytogenes ATCC ® 19115 ™ B-A70 2.105.5 Pseudomonas aeruginosa ATCC ® 27853 ™ B-A71 2.105.5 Salmonella enterica subsp. enterica serovar B-A72 2.105.5 Enteritidis ATCC ® 13076 ™ Salmonella enterica subsp. enterica serovar B-A73 2.105.5 Typhimurium ATCC ® 13311 ™ Streptococcus pneumoniae ATCC ® 27336 ™ B-A75 2.105.5 Streptococcus egui subsp. zooepidemicus B-A77 2.105.5 ATCC ® 43079 ™ Streptococcus pyogenes ATCC ® BAA-1323 ™ B-B26 2.106.5

List 11-1: extracts from herbal compositions containing: Filipendula ulmaria (PA13), Camellia sinensis (PA21) and at least one among 3: Eugenia caryophyllus (PC20), Vitis vinifera var. tinctoria (PA22), Desmodium adscendens (PB07) used in Example-11:

M[20P]-297: PA10; PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA29; PA33; PA57; PA67; PA69; PB07; PB12; PB60; PC20; PC37; PC49; PC98;

M[20P]-416: PA11; PA13; PA14; PA20; PA21; PA25; PA26; PA28; PA29; PA67; PA75; PB02; PB12; PB27; PB33; PB60; PB88; PC20; PC49; PC60;

M[20P]-418: PA10; PA11; PA12; PA13; PA21; PA22; PA23; PA24; PA26; PA39; PB07; PB12; PB38; PC11; PC12; PC20; PC23; PC24; PC49; PC55;

M[20P]-527: PA10; PA10; PA11; PA13; PA15; PA20; PA20; PA21; PA23; PA26 (3 spoons); PA33; PB08; PB12 (2 spoons); PB60; PB88; PC12; PC20;

M[20P]-557: PA00; PA10; PA20; PA21; PA23; PA24; PA29; PA57; PB07; PB12 (4 spoons); PC11 (2 spoons); PC12; PC20; PC51; PC55; PC67;

M[20P]-573: PA10; PA11; PA13 (3 spoons); PA14; PA18; PA21; PA23; PA29; PA67; PB00; PB07; PB12; PB33; PB77; PC11; PC52; PC60; PC71;

M[20P]-576: PA11 (2 spoons); PA12; PA13 (3 spoons); PA14 (2 spoons); PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37; PC49;

M[19P]-576: PA11 (2 spoons); PA12; PA13 (3 spoons); PA14 (2 spoons); PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37;

M[5P]-1: PA12; PA13; PA21; PA22; PB12;

M[5P]-2: PA11; PA13; PA21; PA22; PB12;

For the mixes M[5P]-1, M[5P]-2 and M[19P]-576, the dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France). For the other mixes, the dried plants of pharmaceutical grade were obtained from “Pharmacie Fontgiève” (Clermont-Ferrand, France, plants PA00 to PB12) and “Pharmacie St Herem” (Clermont-Ferrand, France, plants PB13 to PD02).

All herbal compositions of 20 plants were prepared according to the previously described Method A.

Starting from the herbal compositions, corresponding water extracts (or processed samples) were prepared according to the previously described Method B using 40 mL of water containing 100 g/l of sucrose for water extraction and including the decantation step before filtration.

Herbal compositions M[19P]-576, M[5P]-1 and M[5P]-2 were prepared by weighting each dried herb as indicated in Table 11-2

TABLE 11-2 weight of dried plant (mg) used in the herbal compositions of M[19P]-576, M[5P]-1 and M[5P]-2 used in Example-11 PA11 PA12 PA13 PA14 PA20 PA21 PA22 PA29 PA39 PB12 PB60 PC20 PC26 PC33 PC37 M[19P]- 409 145 573 316 277 271 207 380 207 247 206 236 198 187 154 576 M[5P]-1 903 1574 2237 1263 2022 M[5P]-2 756 720 1027 577 925

Starting from the herbal compositions, corresponding water extracts (or processed samples) were prepared according to the previously described Method B using, using, respectively, 30 mL, 40 mL and 40 mL, of water without sucrose with, respectively, M[19P]-576, M[5P]-1 and M[5P]2, for water extraction and including the decantation step before filtration.

The biological activity of each processed sample was determined according to the previously described Method C by using the following strains at the indicated final dilutions of processed samples is given in Table 11-3.

The microbial planktonic growth inhibition percentage average is given in Table 11-3.

TABLE 11-3 planktonic growth inhibition percentage on the 14 tested microbial strains of the processed sample of composition disclosed in List 11-1 M[20C]- M[20C]- M[20C]- M[20C]- M[20C]- M[20C]- M[20C]- M[19C]- 297 416 418 527 557 573 576 576 M[5P]-1 M[5P]-2 Dilution 1:10 1:10 1:10 1:10 1:10 1:10 1:10 1:20 1:20 1:20 B-A49 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% B-A08 100%  99% 100% 100% 100%  98%  98% 100% 100% 100% B-A09  89%  97%  98%  99%  99% 100% 100% 100% 100% 100% B-A61  25%  69%  36%  45%  51%  55%  90% ND ND ND B-A67  45%  52%  59%  66%  62%  52%  39%  34%  55%  38% B-A68 100%  97% 100% 100% 100%  94%  92% 100% 100% 100% B-A69  −1%  21%  21%  38%  30%  30%  −1%  38% 100%  38% B-A70  39%  52%  53%  74%  54%  57%  32%  34% 100%  40% B-A71  63%  71%  69%  75%  75%  82%  71%  42% 100%  55% B-A72  47%  53%  59%  63%  58%  58%  32%  39%  62%  42% B-A73  59%  59%  59%  71%  72%  58%  32%  42% 100%  45% B-A75  51%  52%  63%  71%  63%  57%  31% ND ND ND B-A77  40%  47%  64%  68%  59%  58%  40% 100% 100% 100% B-B26  58%  64%  72%  70%  74%  42%  93% ND ND ND Note: ″ND″ stands for ″not determined″

Example 12

In this Example, we illustrate antimicrobial activities of a plant mix where the extraction step was realized with the non-aqueous solvents acetone, ethyl-acetate, ethanol, used alone or as binary mixes as indicated in Table 12-1. A comparison with water is provided.

TABLE 12-1 composition of the binary mixes used in Example 12 for non-aqueous extraction A) B) C) Ethyl- Ethyl- Acetone acetate acetate Binary to Binary to Binary to solvant Ethanol solvant Ethanol solvant Acetone name ratio name ratio name ratio AC9ET1 90/10 EA9ET1 90/10 EA9AC1 90/10 AC8ET2 80/20 EA8ET2 80/20 EA8AC2 80/20 AC7ET3 70/30 EA7ET3 70/30 EA7AC3 70/30 AC6ET4 60/40 EA6ET4 60/40 EA6AC4 60/40 AC5ET5 50/50 EA5ET5 50/50 EA5AC5 50/50 AC4ET6 40/60 EA4ET6 40/60 EA4AC6 40/60 AC3ET7 30/70 EA3ET7 30/70 EA3AC7 30/70 AC2ET8 20/80 EA2ET8 20/80 EA2AC8 20/80 AC1ET9 10/90 EA1ET9 10/90 EA1AC9 10/90

The M[20P]-576 dried plant mix was composed of following Table 12-2 and placed in a 500 mL vial and thoroughly mixed by manual flipping and rolling. The dried herbs were obtained form “Herboristerie Cailleau” (Chemillé, France), except PC49 which was obtained from Siccarapam (Aubiat, France).

TABLE 12-2 list of dried plant composing the M[20P]-576 used in Example 12 Dried plant PA11 PA12 PA13 PA14 PA20 PA21 PA22 PA29 PA39 PB12 PB60 PC20 PC26 PC33 PC37 PC49 Weight 29.7 10.5 41.5 22.9 19.8 19.9 15.0 27.5 15.0 17.8 15.0 17.1 14.4 13.6 11.3 9.3 (g)

For each solvent tested, 5 g of dried plant mix were placed into a 100 ml coloured vial (Schott, Germany) with a magnetic bar and 50 ml solvent was added. The liquid suspension was stirred with a magnetic stirrer (RO 15P IKA, Germany) at maximum speed during 48 hours at room temperature. Each suspension/solution was filtered by gravity using a 250 mm folded filter (J025106, Prats Dumas, France) placed on a glass funnel (Schott, Germany) itself placed into an evaporation flask. The flask containing the liquid which crossed the filter is placed on a rotary vacuum evaporation device (Rotavapor® R205+V800 vacuum controller, Büchi, Switzerland) and evaporation is performed at 35° C. with vacuum adjusted to be below the solvent's ebullition point, until all solvent has evaporated. The remaining material is weighted and dissolved into DMSO (Acros organic, USA) qsp 10 g/L to form the extracted mix.

Two water extracts were prepared according to the previously described Method B, where the spoons of dried plants were replaced by 2 g of the M[20P]-576 dried plant mix of Example 12, using 40 mL of water for water extraction and including the decantation step before filtration. The dry residue content of one of the plant mix extract has been determined by weighting the lyophilisation residue obtained with a lyophilisation device (FreeZone™ 2.5, Labconco USA, with RZ6 Vacuubrand, Germany) of the frozen extract.

These dry residue contents were used to deduce the specific activities of the different extracts.

The biological activity of each extracted mix was determined according to the previously described Method C by using the strains indicted in Table 12-3, where the dilution of the extracted mix was: 1:20, 1:40, 1:80, 1:160. The minimal inhibiting concentration (MIC) was noted by visual observation of the wells as the lowest concentration where no growth is observed.

TABLE 12-3 list of the bacterial strains used in the Example 12 Staphylococcus aureus Staphylococcus epidermidis NCTC 12493 ™ B-A47 ATCC ® 12228 ™ B-A22 ATCC ® 33591 ™ B-A48 ATCC ® 700296 ™ B-A23 ATCC ® 33592 ™ B-A49 ATCC ® 49461 ™ B-A24 NCTC 43300 ™ B-A51 ATCC ® 14990 ™ B-A08

The results are compiled in tables 12-4 for the different solvents.

TABLE 12-4 CMI in mg/L of the extracted mixes using, A) acetone, ethanol-acetate, ethanol and water as a solvent, B), different proportions of acetone and ethanol as binary solvent as indicated in Table 12-2-A, C) different proportions of ethanol-acetate and ethanol as binary solvent as indicated in Table 12-2-B, and D) using different proportions of ethanol-acetate and acetone as binary solvent as indicated in Table 12-2-C. A) Acetone Ethanol-Acetate Ethanol Water B-A47 500 500 500 725 B-A48 500 500 500 725 B-A49 500 500 500 725 B-A51 500 500 500 725 B-A22 250 250 250 363 B-A23 250 250 250 363 B-A24 250 250 250 363 B-A08 250 250 250 725 B) AC9ET1 AC8ET2 AC7ET3 AC6ET4 AC5ET5 AC4ET6 AC3ET7 AC2ET8 AC1ET9 B-A47 500 500 500 500 500 500 500 500 500 B-A48 250 250 250 250 250 500 500 500 500 B-A49 250 250 250 250 250 500 500 500 500 B-A51 500 500 500 500 500 500 500 500 500 B-A22 250 250 250 250 250 250 250 250 250 B-A23 250 125 125 125 125 250 250 250 250 B-A24 250 250 250 250 250 250 250 250 250 B-A08 250 250 250 250 250 250 250 250 250 C) EA9ET1 EA8ET2 EA7ET3 EA6ET4 EA5ET5 EA4ET6 EA3ET7 EA2ET8 EA1ET9 B-A47 500 500 500 500 500 500 500 500 500 B-A48 250 250 250 250 250 500 500 500 500 B-A49 250 250 250 250 250 500 500 500 500 B-A51 250 500 500 500 500 500 500 500 500 B-A22 250 250 250 250 250 250 250 250 250 B-A23 250 250 250 250 250 250 250 250 250 B-A24 250 250 250 250 250 250 250 250 250 B-A08 250 250 250 250 250 250 250 250 250 D) EA9AC1 EA8AC2 EA7AC3 EA6AC4 EA5AC5 EA4AC6 EA3AC7 EA2AC8 EA1AC9 B-A47 500 500 500 500 500 500 500 500 500 B-A48 500 500 500 500 500 500 500 500 500 B-A49 500 500 500 500 500 500 500 500 500 B-A51 500 500 500 500 500 500 500 500 500 B-A22 500 250 250 250 250 250 250 250 250 B-A23 500 250 250 250 250 250 250 250 250 B-A24 500 250 250 250 250 250 250 250 250 B-A08 500 500 250 250 250 250 250 250 250

Example 13

In this Example, we illustrate antimicrobial activities of a plant mixes prepared according to the previously described Method B, where the added carbohydrate is chosen among sucrose, dextrose, maltose and galactose.

The M[19P]-576, M [5P]-1 and M[5P]-2 dried plant mix were composed following Table 13-1 and placed in a 500 mL vial and thoroughly mixed by manual flipping and rolling. The dried herbs were obtained form “Herboristerie Cailleau” (Chemillé, France), except PC49 which was obtained from Siccarapam (Aubiat, France).

TABLE 13-1 list of dried plant composing: A) the M[20P]-576 mix, B) the M[5P]-1 mix and C), the M[5P]-2 mix used in Example 13 A) Dried plant PA11 PA12 PA13 PA14 PA20 PA21 PA22 PA29 PA39 PB12 PB60 PC20 PC26 PC33 PC37 PC49 Weight 29.7 10.5 41.5 22.9 19.8 19.9 15.0 27.5 15.0 17.8 15.0 17.1 14.4 13.6 11.3 9.3 (g) B) C) Dried Dried plant PA12 PA13 PA21 PA22 PB12 plant PA11 PA13 PA21 PA22 PB12 Weight 34.1 59.2 84.1 47.5 75.8 Weight 56.9 54 76.9 43.4 69.4 (g) (g)

For each dried plant mix, extracts were prepared according to the previously described Method B, where the spoons of dried plants were replaced by 2 g of the plant mix of Example 13, using 40 mL of water and the amounts of carbohydrate indicated in Table13-3 for water extraction and including the decantation step before filtration.

The dry residue content of the plant mixes without carbohydrate was determined separately, by weighting the lyophilisation residue obtained with a lyophilisation device (FreeZone™ 2.5, Labconco USA, with RZ6 Vacuubrand, Germany) of a frozen 40 mL extract prepared according to the previously described Method B, where the spoons of dried plants were replaced by 2 g of the plant mix of Example 13, using 40 mL of water for water extraction and including the decantation step before filtration. These dry residue contents were used to deduce the specific activities of the different extracts, where the carbohydrate is considered as an adjuvant and not accounted as part of the active ingredient.

The biological activity of each extracted mix was determined according to the previously described Method C by using the strains indicted in Table 13-2, where the dilution of the extracted mix was: 1:20, 1:40, 1:80, 1:160. The minimal inhibiting concentration (MIC) was noted by visual observation of the wells as the lowest concentration where no growth is observed.

TABLE 13-2 list of the bacterial strains used in the Example 13 Staphylococcus aureus Staphylococcus epidermidis NCTC 12493 ™ B-A47 ATCC ® 12228 ™ B-A22 ATCC ® 33591 ™ B-A48 ATCC ® 700296 ™ B-A23 ATCC ® 33592 ™ B-A49 ATCC ® 49461 ™ B-A24 NCTC 43300 ™ B-A51 ATCC ® 14990 ™ B-A08

The results are compiled in tables 13-4.

TABLE 13-3 MIC in mg/L of the extracted mixes using sucrose, lactose, dextrose, maltose or galactose for: A) the M[20P]-576 mix, B) the M[5P]-1 mix and C), the M[5P]-2 mix A) Sucrose Lactose Dextrose Maltose Galactose M[20P]-576 100 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L B-A47 725 >725 >725 725 >725 >725 >725 >725 725 725 725 725 725 B-A48 725 >725 >725 725 >725 >725 >725 725 725 725 725 725 725 B-A49 725 >725 >725 725 >725 >725 >725 725 725 725 725 725 725 B-A51 725 >725 >725 725 >725 >725 >725 725 725 725 725 725 725 B-A22 363 725 725 725 725 725 363 363 363 363 363 363 363 B-A23 363 725 725 363 725 725 363 363 363 363 363 363 363 B-A24 363 725 725 725 725 725 363 363 363 363 363 363 363 B-A08 725 725 725 725 725 725 725 363 363 363 363 363 363 B) Sucrose Lactose Dextrose Maltose Galactose M[5P]-1 100 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L B-A47 327 327 327 327 327 327 327 327 327 327 327 327 327 B-A48 327 327 327 327 327 327 327 327 327 327 327 327 327 B-A49 327 327 327 327 327 327 327 327 327 327 327 327 653 B-A47 327 327 327 327 327 327 327 327 327 327 327 327 327 B-A22 327 327 327 327 327 327 327 163 163 163 163 327 327 B-A23 163 163 327 163 163 163 327 163 163 163 163 327 163 B-A24 327 327 327 327 327 327 327 163 163 163 163 327 163 B-A08 327 327 327 327 327 327 327 163 327 327 163 327 327 C) Sucrose Lactose Dextrose Maltose Galactose M[5P]-2 100 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L 100 g/L 50 g/L 25 g/L B-A47 344 344 344 344 344 344 344 344 344 344 344 344 688 B-A48 344 344 344 344 344 344 344 344 344 344 344 344 344 B-A49 344 344 344 344 344 344 344 344 344 344 344 344 688 B-A51 344 344 344 344 344 344 344 688 344 688 344 344 688 B-A22 344 344 344 344 344 344 344 344 344 344 344 344 344 B-A23 344 344 344 344 344 344 344 344 344 344 344 344 344 B-A24 344 344 344 344 344 344 344 344 344 344 344 344 344 B-A08 344 344 344 344 344 344 344 344 344 344 344 344 344

Example 14

In this Example, we illustrate the antimicrobial activity enhancement of the M[20P]-576 herbal mix extract towards commonly used antibiotics.

TABLE 14-1 bacterial species and strains used in Example 14 Strain full name Strain code Staphylococcus aureus subsp. aureus ATCC ® 25923 ™ B-A17 Staphylococcus aureus subsp. aureus ATCC ® 29213 ™ B-A21

List 14-1: herbal compositions of processed sample used in Example 14:

M[19P]-576: PA11; PA12; PA13; PA14; PA20; PA21; PA22; PA29; PA39; PB12; PB60; PC20; PC26; PC33; PC37.

The dried plants of pharmaceutical grade were obtained from “Herboristerie Cailleau” (Chemillé, France).

Herbal compositions M[19P]-576 was prepared by weighting each dried herb as indicated in Table 14-2

TABLE 14-2 weight of dried plant (mg) used in the herbal compositions of M[19P]-576 PA11 PA12 PA13 PA14 PA20 PA21 PA22 PA29 PA39 PB12 PB60 PC20 PC26 PC33 PC37 M[19P]-576 409 145 573 316 277 271 207 380 207 247 206 236 198 187 154

Starting from the herbal composition, the corresponding water extracts (or processed samples) was prepared according to the previously described Method B using 2.06 g of the above herbal composition with 40 mL water with 100 g/L sucrose for water extraction and including the decantation step before filtration. In an independent water extract preparation without sucrose, it has been measured that 2.06 g/40 mL of herbal composition corresponds to 14.5 g/L as deduced from the weight of the lyophilized dry herbal extract.

The biological activity of each processed sample was determined according to the previously described Method C by using the following strains at the indicated final dilutions of processed samples is given in Table 14-3. In Example 14 we calculate the growth of the bacteria in presence of antibiotic and/or herbal mix relative to the growth in the absence of both antibiotic and herbal mix extract.

The bacteria planktonic growth inhibition percentage average is given in Table 14-3.

It has to be noted that the presence of the herbal extract M[19P]-576 has three effects at the concentration used: 1) it reduces the growth of the bacteria at low concentration and absence of antibiotic, 2) it reduces the increase of bacterial growth induced by low antibiotic concentration relative to the growth in the absence of antibiotics, and 3) it reduces the minimal inhibitory concentration (MIC), of, respectively, vancomycin for B-A17 and of ampicillin and penicillin for B-A21, at M[20C]-576 concentration starting at, respectively, 90.6 μg/mL, 181.3 μg/mL, and 90.6 μg/mL.

TABLE 14-3 planktonic growth in the presence of different extract concentrations and anttibiotic concentrations relative to the grrowth of the bacteria in the absence of both extract and antibiotic, on two different strains M[20C]- 576 Antibiotic concentration (μg/mL) Strain (μg/mL) 0 3.91 7.81 15.63 31.25 62.50 125 250 500 1000 2000 4000 Ampicillin B-A17 0 100% 176% 176% 148% 131% 112%   2%   0%   0%   0%  0% −1% B-A21 90.6  57%  64%  76%  75%  58%  18%   1%   0%   0%   0%  0%  0% 181.3  54%  52%  53%  52%  31%   7%  −4%  −4%  −4%  −4% −5% −1% 0 100% 153% 132% 131% 132% 116% 150% 113% 112% 106% −1%  0% 90.6  64% 108%  83%  82%  82%  74%  83%  75%  83%   0%  0%  0% 181.3  42%  44%  63%  70%  69%  70%  62%  62%  61%  −2% −3% −2% Penicillin B-A17 0 100% 137% 138%   3%   1%   0%  −1%   0%   0%   0%  0%  0% B-A21 90.6  53%  39%  25%   1%   0%   0%   0%   0%   0%   0%  0%  0% 181.3  51%  23%   5%  −3%  −3%  −3%  −3%  −2%  −3%  −4% −4% −2% 0 100% 135% 116% 115% 113% 108% 106% 101%   0%   0%  0%  0% 90.6  65% 104%  83%  83%  84%  80%  74%  26%   0%   0%  0%  0% 181.3  44%  62%  43%  70%  61%  60%  45%  −3%  −3%  −3% −3% −3% Vancomycin B-A17 0 100% 135% 118% 114% 117% 115% 116% 118% 128% 117%  0%  0% B-A21 90.6  62%  65%  66%  65%  74%  70%  67%  66%  63%   0%  0%  0% 181.3  42%  40%  41%  53%  55%  55%  54%  46%  47%  −3% −3% −2% 0 100% 118% 117% 113% 117% 110% 102% 102% 102%   0%  0%  0% 90.6  45% 102%  82%  88%  77%  76%  77%  74%  67%   0%  0%  0% 181.3  58%  48%  63%  57%  60%  57%  41%  57%  32%  −2% −3%  0%

Example 15

In this Example, we illustrate how to adapt the temperature in Method B in order to cope with industrial constraints.

The dried plants of organic grade were obtained from “Herboristerie Cailleau” (Chemillé, France).

Herbal compositions M[5P]-1 was prepared by weighting each dried herb as indicated in Table 15-1

TABLE 15-1 weight of dried plant (mg) used in the herbal compositions of M[5P]-1 PA12 PA13 PA21 PA22 PB12 M[5P]-1 395.2 688.8 978.9 552.5 884.6

A first extract M[5P]-1-A was prepared by incorporating 120 g of the herbal composition into 600 mL of water contained in a 2 L beaker (Schott, Germany). A second extract M[5P]-1-B was prepared by placing 120 g of the herbal composition into a 25×30 cm organic grade cotton bag (Ecobags, USA), and the filled bag was placed into 600 mL of water contained into a 2 L beaker (Schott, Germany). A third extract M[5P]-1-C was prepared by twice placing 60 g of the herbal composition into a 25×30 cm organic grade cotton bag (Ecobags, USA), and the two filled bags were placed into 600 mL of water contained into a single 2 L beaker (Schott, Germany). Each, respectively, M[5P]-1-A, M[5P]-1-B and M[5P]-1-C, preparation was left for maceration for 3 hours at room temperature and then placed in its beaker into an autoclave (VWR Vapour Line Eco 25, USA) for 30 minutes at, respectively, 121° C., 121° C. and 134° C. For each of M[5P]-1-B and M[5P]-1-C, the cotton bags were pressed using a manually operated 5 L fruit press (Brouwland, Belgium) and the recovered liquid was placed back into the 2 L beaker. Each beaker was left for decantation and the necessary amount of supernatant liquid was used to measure its biological activity.

TABLE 15-2 bacterial species and strains used in Example 15 Strain full name Strain code Staphylococcus aureus subsp. aureus ATCC ® 25923 ™ B-A17 Staphylococcus aureus subsp. aureus ATCC ® 29213 ™ B-A21 Staphylococcus aureus subsp. aureus ATCC ® 33592 ™ B-A49

The biological activity of each processed sample was determined according to the previously described Method C by using the strains listed in Table 15-2 at final dilutions of 1:40, 1:80, 1:160 and 1:320. The dilution corresponding to the minimal inhibiting concentration (MIC) was noted by visual observation of the wells as the lowest concentration where no growth is observed and is reported into Table 15-3

TABLE 15-3 dilution corresponding to the minimal inhibiting concentration (MIC) of the extracts realized with different industrial details and temperature M[5P]-1-A at 121° M[5P]-1-B at 121° M[5P]-1-C at 134°C B-A17 1/80 1/40 1/80 B-A21 1/80 1/40 1/80 B-A49 1/80 1/40  1/160

It is to be noted that introducing the cotton bag to ease the industrial process reduced slightly the activity of the extract as compared to the unsatisfactory process without bag. Increasing the temperature of the autoclave during the hot extraction phase allowed to recover the full activity.

Example 16

In the following example, the quantitative difference of antimicrobial activities between extracts of mix of plants versus mixing extracts of plants are illustrated.

The extract of the herbal compositions described hereinbelow were prepared according to Method B described on page 15, but without the addition of sucrose as disclosed on page 6.

Specifically, the hereinbelow specified amounts of dried plant powders were dissolved in 20 ml or 21.5 ml of alimentary grade spring water in falcon tubes. Each falcon tube was agitated using a vortex mixer for 10 seconds, incubated at room temperature for 10 minutes and placed in an autoclave for a 20 minutes sterilization cycle.

Extract Mix M[7P]-9 was prepared adding to a falcon tube the following amounts of dried powders in 21.5 ml of water: 273 mg of Rheum palmatum (PA11), 116 mg of Rosmarinus officinalis (PA12), 206 mg of Filipendula ulmaria (PA13), 282 mg of Camellia sinensis (PA21), 206 mg of Vitis vinifera var. tinctoria (PA22), 229 mg of Arctostaphylos uva-ursi (PB12) and 276 mg of Eugenia caryophyllus (PC20). Therefore, the w/w weight ratio between Filipendula ulmaria and Rheum palmatum was 0.7, the w/w weight ratio between Filipendula ulmaria and Rosmarinus officinalis was 1.8, the w/w weight ratio between Filipendula ulmaria and Camellia sinensis was 0.7, the w/w weight ratio between Filipendula ulmaria and Vitis vinifera var. tinctoria was 1, the w/w weight ratio between Filipendula ulmaria and Arctostaphylos uva-ursi was 0.9 and the w/w weight ratio between Filipendula ulmaria and Eugenia caryophyllus was 0.7.

The extraction was performed as described in Method B as discussed above.

The single plant extract EPA11 was prepared adding to a falcon tube the 1652 mg of Rheum palmatum (PA11) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

The single plant extract EPA12 was prepared adding to a falcon tube the 976 mg of Rosmarinus officinalis (PA12) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

The single plant extract EPA13 was prepared adding to a falcon tube the 1244 mg of Filipendula ulmaria (PA13) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

The single plant extract EPA21 was prepared adding to a falcon tube the 1840 mg of Camellia sinensis (PA21) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

The single plant extract EPA22 was prepared adding to a falcon tube the 1295 mg of Vitis vinifera var. tinctoria (PA22) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

The single plant extract EPB12 was prepared adding to a falcon tube the 1506 mg of Vitis vinifera var. tinctoria (PB12) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

The single plant extract EPC20 was prepared adding to a falcon tube the 1711 mg of Eugenia caryophyllus (PC20) in 20 ml of water. The extraction was otherwise performed as described in Method B as discussed above.

Mix M[7E]-9 was prepared by mixing extracts EPA11, EPA12, EPA13, EPA21, EPA22, EPB12 and EPC20 in such proportions as to obtain a mix nominally equivalent in its composition in dried plants to M[7P]-9. This condition is obtained by using for each botanical subscribed k (k among A11, A12, A13, A21, A22, B12 and C20) a volume VEPk such that SVEP×QMk/21.5 mL=VEPk×QEPk/20 mL, where SVEP is the sum of all VEPk, QMk is the amount of dry plant k used to prepare Mix M[7E]-9 and QEk is the amount of dry plant k used to prepare EPk. Mix M[7E]-9 was thus prepared by mixing 157 μL of Rheum palmatum EPA11, 113 μL of Rosmarinus officinalis EPA12, 157 μL of Filipendula ulmaria EPA13, 146 μL of Camellia sinensis EPA21, 151 μL of Vitis vinifera var. tinctoria EPA22, 145 μL of Arctostaphylos uva-ursi EPB12 and 153 μL of Eugenia caryophyllus EPC20.

For each extract the biological activity was determined according to the previously described Method C, with the following final dilutions of processed samples: 1:10 (Pseudomonas aeruginosa ATCC® 27853™), and 1:40 (Staphylococcus aureus subsp. aureus ATCC® 29213™). Optical density measures are taken every hour and reported in the following graphs which show the growth kinetics of Pseudomonas aeruginosa (first and second graphs) and Staphylococcus aureus (third and fourth graphs) without and with mixes Mix M[7P]-9 and M[7E]-9.

From the results reported in FIGS. 1-4, it is evident that the biological activity of the growth kinetics of Pseudomonas aeruginosa (FIGS. 1 and 2) and Staphylococcus aureus (FIGS. 3 and 4) is significantly decreased in the presence of the extract of herbal compositions presently claimed (Mix M[7P]-9) compared to the growth achieved with M[7E]-9, i.e., the corresponding composition of extracts, or in the absence of any mix, i.e., the control. Thus, there are quantitative and qualitative differences between the herbal compositions presently claimed and the composition of the corresponding natural herbal extracts. That is, the effects obtainable with the claimed invention are markedly different from the effects obtainable from its closest naturally occurring counterparts.

Example 17

In the following example we report the composition in dry plants of some final products prepared according to the invention.

For each of the following final products we report in a corresponding table the amount of dry plant (in grams) used for 1 kg of ingredient and the amount of extract (in grams) in 1 kg of ingredient, where “ingredient” means the final extract.

To calculate the amount of extract of single plant, we started with a separate test experiment for each plant “i” taken individually to get Xi=E′i/D′i, where E′ is extract and D′ dry plant, both measured.

Then, for the extract from the MIX of plants, we calculated Ei=Xi, Di, Et/sum(Xi. Di), where Ei, is a calcualted amount and represents the “theoretical” extract and Di (the measured weight) is the dry plant and where Et is the experimentally measured extract obtained from sum(Di) amount of mix.

1. Ingredient of a Cosmetic Final Product A Having Antibacterial Activity:

TABLE 17.1.1 Composition in dry plants for product A Amount of dry Amount of w/w weight ratio plant (g) used extract (g) Filipendula Botanical for 1 kg of in 1 kg of ulmaria/other Code Name ingredient ingredient dry plant PA12 Rosmarinus 30 g 2.8 g 1.8 officinalis L. PA13 Filipendula 53 g 3.9 g 1 ulmaria (L.) Maxim. PA21 Camellia 75 g 8.3 g 0.7 sinensis (L.) Kuntze PA22 Vitis vinifera 42 g 4.0 g 1.3 L.

TABLE 17.1.2 Ingredient used in final product A at recommended proportion of 5% to 20%. Range of amount of dry Range of amount of Botanical plant (g) used for 1 kg of extract (g) in 1 kg of Code Name final product final product PA12 Rosmarinus 1.5 g-6 g   0.1 g-0.6 g officinalis L. PA13 Filipendula  2.6 g-10.5 g 0.2 g-0.8 g ulmaria (L.) Maxim. PA21 Camellia 3.7 g-15 g  0.4 g-1.7 g sinensis (L.) Kuntze PA22 Vitis vinifera 2.1 g-8.5 g 0.2 g-0.8 g L.

TABLE 17.1.3 Final product A typically sold in packages having volumes in the range of 50 mL to 200 mL Range of amount of Range of amount dry plant (g) used for of extract (g) in Botanical 50 mL to 200 mL 50 mL to 200 mL Code Name of final product of final product PA12 Rosmarinus 0.1 g-1.2 g 0.01 g-0.1 g officinalis L. PA13 Filipendula 0.1 g -2.1 g 0.01 g-0.2 g ulmaria (L.) Maxim. PA21 Camellia 0.2 g-3 g   0.02 g-0.3 g sinensis (L.) Kuntze PA22 Vitis vinifera 0.1 g-1.7 g 0.01 g-0.2 g L.

2. Ingredient of a Cosmetic Final Product B Having Antibacterial Activity:

TABLE 17.2.1 Composition in dry plants for product B Amount of Amount of w/w weight ratio dry plant (g) extract (g) Filipendula used for 1 kg in 1 kg of ulmaria/other Code Botanical Name of ingredient ingredient dry plant PA12 Rosmarinus 23 g 3.2 g 1.7 officinalis L. PA13 Filipendula 39 g 4.6 g 1 ulmaria (L.) Maxim. PA21 Camellia 56 g 9.7 g 0.7 sinensis (L.) Kuntze PA22 Vitis vinifera L. 32 g 4.7 g 1.2 PB12 Arctostaphylos 51 g 8.2 g 0.8 uva-ursi(L.) Spreng.

TABLE 17.2.2 Ingredient used in final product B at recommended proportion of 5% to 20%. Range of amount of Range of amount of dry plant (g) used for extract (g) in 1 kg of Code Botanical Name 1 kg of final product final product PA12 Rosmarinus 1.1 g-4.5 g 0.2 g-0.6 g officinalis L. PA13 Filipendula   2 g-7.9 g 0.2 g-0.9 g ulmaria (L.) Maxim. PA21 Camellia sinensis  2.8 g-11.2 g 0.5 g-1.9 g (L.) Kuntze PA22 Vitis vinifera L. 1.6 g-6.3 g 0.2 g-0.9 g PB12 Arctostaphylos  2.5 g-10.1 g 0.4 g-1.6 g uva-ursi(L.) Spreng.

TABLE 17.2.3 Final product B typically sold in packages having volumes in the range of 50 mL to 200 mL Range of amount of Range of amount dry plant (g) used for of extract (g) in 50 mL to 200 mL 50 mL to 200 mL Code Botanical Name of final product of final product PA12 Rosmarinus 0.1 g-0.9 g 0.01 g-0.1 g officinalis L. PA13 Filipendula 0.1 g-1.6 g 0.01 g-0.2 g ulmaria (L.) Maxim. PA21 Camellia sinensis 0.1 g-2.2 g 0.02 g-0.4 g (L.) Kuntze PA22 Vitis vinifera L. 0.1 g-1.3 g 0.01 g-0.2 g PB12 Arctostaphylos 0.1 g-2 g   0.02 g-0.3 g uva-ursi(L.) Spreng.

3. Ingredient of a Cosmetic Final Product C Having Antibacterial Activity:

TABLE 17.3.1 Composition in dry plants for product C Amount of Amount of w/w weight ratio dry plant (g) extract (g) Filipendula used for 1 kg in 1 kg of ulmaria/other Code Botanical Name of ingredient ingredient dry plant PA11 Rheum palmatum 23 g 2.9 g 1.7 L. PA13 Filipendula 39 g   3 g 1 ulmaria (L.) Maxim. PA21 Camellia sinensis 56 g 6.4 g 0.7 (L.) Kuntze PA22 Vitis vinifera var. 32 g 3.1 g 1.2 tinctoria PB12 Arctostaphylos 51 g 5.4 g 0.8 uva-ursi(L.) Spreng.

TABLE 17.3.2 Ingredient used in final product C at recommended proportion of 5% to 20%. Range of amount Range of amount of dry plant (g) of extract used for 1 kg of (g) in 1 kg of Code Botanical Name final product final product PA11 Rheum palmatum 1.1 g-4.5 g 0.1 g-0.6 g L. PA13 Filipendula   2 g-7.9 g 0.2 g-0.6 g ulmaria (L.) Maxim. PA21 Camellia sinensis  2.8 g-11.2 g 0.3 g-1.3 g (L.) Kuntze PA22 Vitis vinifera var. 1.6 g-6.3 g 0.2 g-0.6 g tinctoria PB12 Arctostaphylos  2.5 g-10.1 g 0.3 g-1.1 g uva-ursi(L.) Spreng.

TABLE 17.3.3 Final product C typically sold in packages having volumes in the range of 50 mL to 200 mL Range of amount of Range of amount of dry plant (g) used for extract (g) in 50 mL 50 mL to 200 mL to 200 mL of final Code Botanical Name of final product product PA11 Rheum palmatum 0.1 g-0.9 g 0.01 g-0.1 g L. PA13 Filipendula 0.1 g-1.6 g 0.01 g-0.1 g ulmaria (L.) Maxim. PA21 Camellia sinensis 0.1 g-2.2 g 0.02 g-0.3 g (L.) Kuntze PA22 Vitis vinifera var. 0.1 g-1.3 g 0.01 g-0.1 g tinctoria PB12 Arctostaphylos 0.1 g-2 g   0.01 g-0.2 g uva-ursi(L.) Spreng.

4. Ingredient of a Cosmetic Final Product D Having Antibacterial Activity:

Product D is commercialized as a 50 mL lotion, 150 mL lotion, 200 mL shampoo and 150 mL foam.

TABLE 17.4.1 Composition in dry plants for product D-lotion Amount of dry plant Amount of dry extract used for 50 mL to used for 50 mL to Code Botanical Name 150 mL of lotion 150 mL of lotion PA11 Rheum palmatum L. 282 mg-847 mg 89 mg-268 mg PA12 Rosmarinus 100 mg-300 mg 24 mg-71 mg  officinalis L. PA13 Filipendula ulmaria  396 mg-1188 mg 83 mg-248 mg (L.) Maxim. PA14 Satureja montana L. 150 mg-451 mg 44 mg-133 mg PA20 Valeriana officinalis 187 mg-562 mg 80 mg-241 mg L. PA21 Camellia sinensis (L.) 151 mg-452 mg 44 mg-133 mg Kuntze PA22 Vitis vinifera var. 142 mg-425 mg 33 mg-100 mg tinctoria PA29 Fucus vesiculosus L. 260 mg-780 mg 80 mg-241 mg PA39 Foeniculum vulgare 143 mg-428 mg 36 mg-107 mg Mill. PB12 Arctostaphylos uva- 170 mg-509 mg 45 mg-134 mg ursi (L.) Spreng. PB60 Arbutus unedo L. 144 mg-431 mg 44 mg-132 mg PC20 Syzygium 150 mg-451 mg 44 mg-133 mg aromaticum(L.) Merr. & L. M. Perry PC26 Juniperus communis 135 mg-405 mg 16 mg-48 mg  L. PC33 Combretum 130 mg-389 mg 33 mg-98 mg  micranthum G. Don PC37 Aloysia citriodora 106 mg-318 mg 29 mg-87 mg  Palau PF38 Tanacetum  88 mg-263 mg 26 mg-78 mg  parthenium(L.) Sch. Bip.

TABLE 17.4.2 Composition in dry plants for product D-foam Amount Amount of w/w weight of dry dry extract ratio plant used used for Filipendula for 150 mL 150 mL ulmaria/other Code Botanical Name of foam of foam dry plant PA11 Rheum palmatum L. 85 mg 27 mg 1.4 PA12 Rosmarinus 30 mg  7 mg 4 officinalis L. PA13 Filipendula ulmaria 119 mg  25 mg 1 (L.) Maxim. PA14 Satureja montana L. 45 mg 13 mg 2.7 PA20 Valeriana officinalis 56 mg 24 mg 2.1 L. PA21 Camellia sinensis (L.) 45 mg 13 mg 2.7 Kuntze PA22 Vitis vinifera var. 42 mg 10 mg 2.8 tinctoria PA29 Fucus vesiculosus L. 78 mg 24 mg 1.5 PA39 Foeniculum vulgare 43 mg 11 mg 2.8 Mill. PB12 Arctostaphylos uva- 51 mg 13 mg 2.3 ursi (L.) Spreng. PB60 Arbutus unedo L. 43 mg 13 mg 2.8 PC20 Syzygium 45 mg 13 mg 2.6 aromaticum(L.) Merr. & L. M. Perry PC26 Juniperus communis 40 mg  5 mg 2.3 L. PC33 Combretum 39 mg 10 mg 3 micranthum G. Don PC37 Aloysia citriodora 32 mg  9 mg 3.7 Palau PF38 Tanacetum 26 mg  8 mg 4.6 parthenium(L.) Sch. Bip.

TABLE 17.4.3 Composition in dry plants for product D-shampoo Amount of Amount of w/w weight dry plant dry extract ratio used for used for Filipendula 200 mL of 200 mL of ulmaria/other Code Botanical Name shampoo shampoo dry plant PA11 Rheum palmatum L. 23 mg 7 mg 1.4 PA12 Rosmarinus  8 mg 2 mg 4 officinalis L. PA13 Filipendula ulmaria 32 mg 7 mg 1 (L.) Maxim. PA14 Satureja montana L. 12 mg 4 mg 2.7 PA20 Valeriana officinalis 15 mg 6 mg 2.1 L. PA21 Camellia sinensis (L.) 12 mg 4 mg 2.7 Kuntze PA22 Vitis vinifera var. 11 mg 3 mg 2.9 tinctoria PA29 Fucus vesiculosus L. 21 mg 6 mg 1.5 PA39 Foeniculum vulgare 11 mg 3 mg 2.9 Mill. PB12 Arctostaphylos uva- 14 mg 4 mg 2.3 ursi (L.) Spreng. PB60 Arbutus unedo L. 11 mg 4 mg 2.9 PC20 Syzygium 12 mg 4 mg 2.7 aromaticum(L.) Merr. & L. M. Perry PC26 Juniperus communis 11 mg 1 mg 2.9 L. PC33 Combretum 10 mg 3 mg 3.2 micranthum G. Don PC37 Aloysia citriodora  8 mg 2 mg 4 Palau Tanacetum  7 mg 2 mg 4.6 parthenium(L.) Sch. Bip. Note: PC37 is synonym of Lippia citrodora PC20 is synonum of Eugenia caryophyllus

5. Ingredient of a Cosmetic Final Mroduct E Having Antibacterial Activity:

Product B is a cosmetic gel commercialized in two forms “basic” and “forte”, 50 mL each. It is recommended for dry skin, and it has shown to have antibacterial properties in clinical evaluation.

TABLE 17.5.1 Composition in dry plants for product E Amount of dry plant Amount of dry extract used for 50 mL of used for 50 mL of Code Botanical Name basic and forte basic and forte PA11 Rheum palmatum L. 111 mg-332 mg  27 mg-80 mg PA12 Rosmarinus 39 mg-118 mg  7 mg-21 mg officinalis L. PA13 Filipendula ulmaria 155 mg-466 mg  25 mg-74 mg (L.) Maxim. PA14 Satureja montana L. 59 mg-177 mg 13 mg-40 mg PA20 Valeriana officinalis 74 mg-221 mg 24 mg-72 mg L. PA21 Camellia sinensis (L.) 59 mg-177 mg 13 mg-40 mg Kuntze PA22 Vitis vinifera var. 56 mg-167 mg 10 mg-30 mg tinctoria PA29 Fucus vesiculosus L. 102 mg-306 mg  24 mg-72 mg PA39 Foeniculum vulgare 56 mg-168 mg 11 mg-32 mg Mill. PB12 Arctostaphylos uva- 67 mg-200 mg 13 mg-40 mg ursi (L.) Spreng. PB60 Arbutus unedo L. 56 mg-169 mg 13 mg-39 mg PC20 Syzygium 59 mg-177 mg 13 mg-40 mg aromaticum(L.) Merr. & L. M. Perry PC26 Juniperus communis 53 mg-159 mg  5 mg-14 mg L. PC33 Combretum 51 mg-153 mg 10 mg-29 mg micranthum G. Don PC37 Aloysia citriodora 42 mg-125 mg  9 mg-26 mg Palau Tanacetum 34 mg-103 mg  8 mg-23 mg parthenium(L.) Sch. Bip. Coffea canephora 108 mg-323 mg  27 mg-80 mg Pierre Ex. Froehner Note: PC37 is synonym of Lippia citrodora PC20 is synonym of Eugenia caryophyllus

6. Ingredient of a Drug Final Product G Having Antibacterial Activity:

Product G is a drug for oral administration to adult human as an alternative to antibiotic, typically 5 g of extract per dose to be administrated 1 to 4 times daily. It has shown to have antibacterial properties.

TABLE 17.6.1 Composition in dry plants for product G for human treatment Amount of Amount w/w weight ratio dry plant of extract Filipendula (g) used for (g) in 1 ulmaria/other Code Botanical Name one dose one dose dry plant PA12 Rosmarinus 2.3 g 0.5 g 1.7 officinalis L. PA13 Filipendula 3.9 g 0.8 g 1 ulmaria (L.) Maxim. PA21 Camellia sinensis 5.6 g 1.6 g 0.7 (L.) Kuntze PA22 Vitis vinifera var. 3.2 g 0.8 g 1.2 tinctoria PB12 Arctostaphylos 5.0 g 1.4 g 0.8 uva-ursi (L.) Spreng.

Claims

1) An extract of an herbal composition comprising the following dried plants: Filipendula ulmaria, Camellia sinensis and Vitis vinifera var. tinctoria, wherein the w/w weight ratio between Filipendula ulmaria and each of the dried Camellia sinensis and Vitis vinifera var. tinctoria is between 0.2 and 5.

2) The extract according to claim 1, wherein the w/w weight ratio between Filipendula ulmaria and each of the dried Camellia sinensis and Vitis vinifera var. tinctoria is between 0.3 and 3.

3) The extract according to claim 1, wherein the w/w weight ratio between Filipendula ulmaria and each of the dried Camellia sinensis and Vitis vinifera var. tinctoria is between 0.5 and 2.5.

4) The extract according to claim 1, wherein the herbal composition further comprises one or more additional dried plants selected among: Achillea millefolium, Acorus calamus, Agrimonia eupatoria, Agropyrum repens, Agropyrum repens, Alchemilla vulgaris, Alkanna tinctoria, Althaea officinalis, Anethum graveolens, Angelica archangelica, Arbutus unedo, Arnica montana, Artemisia pontica, Artemisia vulgaris, Asparagus officinalis, Asparagus officinalis, Asperula odorata, Betula pendula, Borrago officinalis, Buxus sempervirens, Calamintha officinalis, Calendula officinalis, Calluna vulgaris, Carum carvi, Cassia angustifolia, Centaurea cyanus, Centaurium erythraea, Centella asiatica, Cetraria islandica, Chamaemelum nobile, Chamomilla recutita, Chrysanthellum americanum, Cichorium endivia, Cichorium intybus, Cinnamomum zeylanicum, Citrus aurantium, Combretum micranthum, Crataegus oxyacantha, Cuminum cyminum, Cupressus sempervirens, Curcuma zedoaria, Cynara scolymus, Cytisus scoparius, Desmodium adscendens, Elettaria cardamomum, Eleutherococcus senticosus, Epilobium parviflorum, Erysimum officinale, Eucalyptus globulus, Eugenia caryophyllus, Eupatorium cannabinum, Foeniculum vulgare, Fraxinus excelsior, Fucus vesiculosus, Fumaria officinalis, Galium odoratum, Gentiana lutea, Geranium robertianum, Ginkgo biloba, Glechoma hederacea, Glycyrrhiza glabra, Handroanthus impetiginosus, Harpagophytum procumbens, Hieracium pilosella, Humulus lupulus, Hypericum perforatum, Hyssopus officinalis, Illicium verum, Inula helenium, Juglans regia, Juniperus communis, Lamium album, Lavandula angustifolia, Levisticum officinale, Lippia citriodora, Lotus corniculatus, Lythrum salicaria, Malva sylvestris, Marrubium vulgare, Medicago sativa, Melissa officinalis, Mentha x piperita, Morus nigra, Myrtus communis, Olea europaea, Origanum majorana, Panax ginseng, Papaver rhoeas, Parietaria officinalis, Passiflora incarnata, Petroselinum crispum, Peumus boldus, Phaseolus vulgaris, Pimpinella anisum, Plantago lanceolata, Plantago ovata, Potentilla anserina, Quercus robur, Rhamnus frangula, Rheum palmatum, Rosa centifolia, Rosmarinus officinalis, Rubia tinctorum, Rubus idaeus, Salix alba, Salvia officinalis, Sambucus nigra, Satureja montana, Silybum marianum, Solanum dulcamara, Tabebuia impetiginosa, Tanacetum vulgare, Taraxacum officinalis, Thymus serpyllum, Thymus vulgaris, Tilia tomentosa, Tilia cordata, Trigonella foenum-graecum, Tussilago farfara, Vaccinium myrtillus, Valeriana officinalis, Verbascum thapsus, Verbena officinalis, Viscum album, Zea mays and Zingiber officinale.

5) The extract according to claim 4, wherein the herbal composition comprises from three to seven dried plants selected among Filipendula ulmaria, Camellia sinensis, Arctostaphylos uva-ursi, Rheum palmatum, Rosmarinus officinalis, Vitis vinifera tinctoria, Desmodium adscendes, Eugenia caryophyllus and Eucalyptus globulus, wherein at least two of such plants are selected among Filipendula ulmaria, Camellia sinensis and Arctostaphylos uva-ursi and at least one of such plants is selected among Vitis vinifera tinctoria, Desmodium adscendes and Eugenia caryophyllus.

6) The extract according to claim 4, wherein the herbal composition comprises the following dried plants: Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria and Arctostaphylos uva-ursi.

7) The extract according to claim 4, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria and Arctostaphylos uva-ursi.

8) The extract according to claim 4, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria, Eugenia caryophyllus and Arctostaphylos uva-ursi.

9) The extract according to claim 4, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Camellia sinensis, Vitis vinifera tinctoria, Eucalyptus globulus, Arctostaphylos uva-ursi, Mentha spicata and Rubia tinctorium.

10) The extract according to claim 4, wherein the herbal composition comprises at least fourteen or fifteen dried plants selected among the following sixteen: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera tinctoria, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, Lippia citrodora and Tanacetum vulgare.

11) The extract according to claim 4, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera tinctoria, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum and Lippia citrodora.

12) The extract according to claim 4, wherein the herbal composition comprises the following dried plants: Rheum palmatum, Rosmarinus officinalis, Filipendula ulmaria, Satureja montana, Valeriana officinalis, Camellia sinensis, Vitis vinifera tinctoria, Fucus vesiculosus, Foeniculum vulgare, Arctostaphylos uva-ursi, Arbutus unedo, Eugenia caryophyllus, Juniperus communis, Combretum micranthum, Lippia citrodora and Tanacetum vulgare.

13) A food supplement, a nutraceutical, pharmaceutical, veterinary or cosmetic composition or a functional food or a food additive comprising the extract according to claim 4, as active ingredient, together with at least one excipient.

14) The food supplement, the nutraceutical, pharmaceutical, veterinary or cosmetic composition, the functional food, or the food additive according to claim 12, formulated for oral or topic administration.

15) A method of treating, preventing or diagnosing microbial infection with the food supplement, the nutraceutical, pharmaceutical, veterinary or cosmetic composition, the functional food, or the food additive comprising the extract composition according to claim 2, optionally in lyophilized form as active ingredient together with at least one excipient, said method comprising: admininistering to a subject in need thereof a pharmaceutical effective amount of said food supplement, nutraceutical, pharmaceutical, veterinary or cosmetic composition, functional food, or food additive.

16) The method according to claim 15, wherein the infections are caused by Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Streptococcus, Candida or Staphylococcus.

17) The method according to claim 15, wherein the infections are caused by Staphylococcus aureus, Staphylococcus epidermidis or Staphylococcus pseudintermedius.

18) A process for preparing the extract according to claim 4, comprising a) chopping or grinding separately or in a mixture at least two single dried plants to obtain corresponding plant powders; b) mixing at least two different plant powders to obtain an herbal composition; c) adding an extraction solvent to said herbal composition to obtain a corresponding liquid preparation (or extract); d) incubating said liquid preparation at room temperature for a time ranging between 5 and 15 minutes; optionally, e) heating said liquid preparation at a temperature ranging from 60° C. to 134° C. for a time ranging from 5 to 60 minutes; optionally f) centrifuging, collecting the supernatant and filtering the collected supernatant; and, optionally, g) concentrating and/or drying or freeze drying the liquid preparation obtained from step e) or f).

19) The process according to claim 19, where the extraction solvent is water.

20) The process according to claim 20, where the water added in step (c) comprises a sugar dissolved in it at a concentration varying from 1 to 100 g/l, optionally from 50 to 75 g/l.

Patent History
Publication number: 20220354919
Type: Application
Filed: Jul 6, 2022
Publication Date: Nov 10, 2022
Applicant: ALPHANOSOS S.A.S. (Riom)
Inventor: Pascal Mayer (Riom)
Application Number: 17/858,093
Classifications
International Classification: A61K 36/73 (20060101); A61K 36/82 (20060101); A61K 36/87 (20060101); A61K 36/232 (20060101); A61K 36/8965 (20060101); A61K 36/28 (20060101); A61K 36/185 (20060101); A61K 36/35 (20060101); A61K 36/484 (20060101); A61K 36/258 (20060101); A61K 36/537 (20060101); A61K 36/53 (20060101); A61K 36/288 (20060101); A61K 36/45 (20060101); A61K 36/708 (20060101); A61K 36/61 (20060101); A61K 36/74 (20060101); A61P 31/04 (20060101);